Isolation of Zymomonas mobilis Mutant with Increased Tetrahydroxybacteriohopane Content

A mutant of Zymomonas mobilis with an increased content of tetrahydroxybacteriohopane (THBH) was isolated. From comparisons of hopanoids of THBH, a glucosamine and an ether derivative of THBH between the parent strain, THBH-decreased and THBH-increased mutants, the biosynthetic pathway of the side-chain of these hopanoids is discussed.     

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial‐derived liposomes showed that hopanoids protect against several ethanol‐driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter‐lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.     

Quantitative hopanoid analysis enables robust pattern detection and comparison between laboratories

Hopanoids are steroid‐like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2‐methylhopanoid production in Rhodopseudomonas palustris TIE‐1 and purified 2Me‐diplopterol, 2Me‐bacteriohopanetetrol (2Me‐BHT), and their unmethylated species (diplopterol and BHT). We found that 2‐methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me‐diplopterol produces 10× higher ion counts than equivalent quantities of 2Me‐BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC‐MS, however, 2Me‐BHT produces 11× higher ion counts than 2Me‐diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D₄) diplopterol, a precursor for a variety of hopanoids. LC‐MS analysis on a mixture of (D₄)‐diplopterol and phospholipids showed that under the influence of co‐eluted phospholipids, the D4‐diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.     

Lipid remodeling in Rhodopseudomonas palustris TIE‐1 upon loss of hopanoids and hopanoid methylation

The sedimentary record of molecular fossils (biomarkers) can potentially provide important insights into the composition of ancient organisms; however, it only captures a small portion of their original lipid content. To interpret what remains, it is important to consider the potential for functional overlap between different lipids in living cells, and how the presence of one type might impact the abundance of another. Hopanoids are a diverse class of steroid analogs made by bacteria and found in soils, sediments, and sedimentary rocks. Here, we examine the trade‐off between hopanoid production and that of other membrane lipids. We compare lipidomes of the metabolically versatile α‐proteobacterium Rhodopseudomonas palustris TIE‐1 and two hopanoid mutants, detecting native hopanoids simultaneously with other types of polar lipids by electrospray ionization mass spectrometry. In all strains, the phospholipids contain high levels of unsaturated fatty acids (often >80 %). The degree to which unsaturated fatty acids are modified to cyclopropyl fatty acids varies by phospholipid class. Deletion of the capacity for hopanoid production is accompanied by substantive changes to the lipidome, including a several‐fold rise of cardiolipins. Deletion of the ability to make methylated hopanoids has a more subtle effect; however, under photoautotrophic growth conditions, tetrahymanols are upregulated twofold. Together, these results illustrate that the ‘lipid fingerprint’ produced by a micro‐organism can vary depending on the growth condition or loss of single genes, reminding us that the absence of a biomarker does not necessarily imply the absence of a particular source organism.     

Hopanoid production is required for low-pH tolerance, antimicrobial resistance, and motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Δshc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Δshc mutant produced only very small amounts of the hopanoid peak. The Δshc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Δshc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.     

Identification and characterization of Rhodopseudomonas palustris TIE-1 hopanoid biosynthesis mutants

Hopanes preserved in both modern and ancient sediments are recognized as the molecular fossils of bacteriohopanepolyols, pentacyclic hopanoid lipids. Based on the phylogenetic distribution of hopanoid production by extant bacteria, hopanes have been used as indicators of specific bacterial groups and/or their metabolisms. However, our ability to interpret them ultimately depends on understanding the physiological roles of hopanoids in modern bacteria. Toward this end, we set out to identify genes required for hopanoid biosynthesis in the anoxygenic phototroph Rhodopseudomonas palustris TIE-1 to enable selective control of hopanoid production. We attempted to delete 17 genes within a putative hopanoid biosynthetic gene cluster to determine their role, if any, in hopanoid biosynthesis. Two genes, hpnH and hpnG, are required to produce both bacteriohopanetetrol and aminobacteriohopanetriol, whereas a third gene, hpnO, is required only for aminobacteriohopanetriol production. None of the genes in this cluster are required to exclusively synthesize bacteriohopanetetrol, indicating that at least one other hopanoid biosynthesis gene is located elsewhere on the chromosome. Physiological studies with the different deletion mutants demonstrated that unmethylated and C(30) hopanoids are sufficient to maintain cytoplasmic but not outer membrane integrity. These results imply that hopanoid modifications, including methylation of the A-ring and the addition of a polar head group, may have biologic functions beyond playing a role in membrane permeability.     

Diversity of hopanoids and squalene-hopene cyclases across a tropical land-sea gradient

Bacterial hopanoids are ubiquitous in Earth surface environments. They hold promise as environmental and ecological biomarkers, if the phylogeny and physiological drivers of hopanoid biosynthesis can be linked with the distribution of hopanoids observed across a breadth of samples. Here we survey the diversity of hopanoid cyclases from a land‐sea gradient across the island of San Salvador, in the easternmost part of the Bahamas. The distribution of lipids was determined for the same sites, for the first time overlaying quantification of bacteriohopanepolyols with sqhC phylogeny. The results are similar to previous reports: environmental sqhCs average < 65% translated amino acid identity to their closest named relatives, and sequences from putative Proteobacteria dominate. Additionally, a new and apparently ubiquitous group of marine hopanoid producers is identified; it has no identifiable close relatives. The greatest diversity of hopanoid lipids occurs in soil, but hopanoids represent a minor fraction of total soil‐derived lipids. Marine samples contain fewer identifiable hopanoids, but they are more abundant as a fraction of the total extractable lipids. In soil, the dominant compounds are 35‐aminobacteriohopane‐32,33,34‐triol and adenosylhopane. In an upper estuarine sample, bacteriohopanetetrol and 32,35‐anhydrobacteriohopanetetrol dominate; while in lower estuarine and open marine samples, the most abundant are bacteriohopanetetrol and bacteriohopaneribonolactone. Cyclitol ethers are trace components in the soil, absent in the estuary, and of moderate abundance in the open marine setting, suggesting a dominant marine source. Conversely, aminotriol and aminotetrol decrease in abundance or disappear completely from land to ocean, while 2‐methyldiplopterol shows the opposite trend. Small quantities of 2‐methylbacteriohopanepolyols are detectable in all samples. The overall hopanoid distributions may correlate to the major phylogenetic families of hopanoid producers or to the environments in which they are found.     

Hopanoids in marine cyanobacteria: probing their phylogenetic distribution and biological role

Cyanobacteria are key players in the global carbon and nitrogen cycles and are thought to have been responsible for the initial rise of atmospheric oxygen during the Neoarchean. There is evidence that a class of membrane lipids known as hopanoids serve as biomarkers for bacteria, including many cyanobacteria, in the environment and in the geologic record. However, the taxonomic distributions and physiological roles of hopanoids in marine cyanobacteria remain unclear. We examined the distribution of bacteriohopanepolyols (BHPs) in a collection of marine cyanobacterial enrichment and pure cultures and investigated the relationship between the cellular abundance of BHPs and nitrogen limitation in Crocosphaera watsonii, a globally significant nitrogen‐fixing cyanobacterium. In pure culture, BHPs were only detected in species capable of nitrogen fixation, implicating hopanoids as potential markers for diazotrophy in the oceans. The enrichment cultures we examined exhibited a higher degree of BHP diversity, demonstrating that there are presently unaccounted for marine bacteria, possibly cyanobacteria, associated with the production of a range of BHP structures. Crocosphaera watsonii exhibited high membrane hopanoid content consistent with the idea that hopanoids have an important effect on the bulk physical properties of the membrane. However, the abundance of BHPs in C. watsonii did not vary considerably when grown under nitrogen‐limiting and nitrogen‐replete conditions, suggesting that the role of hopanoids in this organism is not directly related to the physiology of nitrogen fixation. Alternatively, we propose that high hopanoid content in C. watsonii may serve to reduce membrane permeability to antimicrobial toxins in the environment.     

Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2)

Streptomyces coelicolor A3(2) contains a cluster of putative isoprenoid and hopanoid biosynthetic genes. The strain does not produce the pentacyclic hopanoids in liquid culture but produces them on solid medium when sporulating. Mutants defective in the formation of aerial mycelium and spores (bld), with the exception of bldB, do not synthesize hopanoids, whereas mutants, which form aerial mycelium but no spores (whi), do. The membrane condensing hopanoids possibly may alleviate stress in aerial mycelium by diminishing water permeability across the membrane.     

Hopanoids play a role in stress tolerance and nutrient storage in the cyanobacterium Nostoc punctiforme

Hopanes are abundant in ancient sedimentary rocks at discrete intervals in Earth history, yet interpreting their significance in the geologic record is complicated by our incomplete knowledge of what their progenitors, hopanoids, do in modern cells. To date, few studies have addressed the breadth of diversity of physiological functions of these lipids and whether those functions are conserved across the hopanoid‐producing bacterial phyla. Here, we generated mutants in the filamentous cyanobacterium, Nostoc punctiforme, that are unable to make all hopanoids (shc) or 2‐methylhopanoids (hpnP). While the absence of hopanoids impedes growth of vegetative cells at high temperature, the shc mutant grows faster at low temperature. This finding is consistent with hopanoids acting as membrane rigidifiers, a function shared by other hopanoid‐producing phyla. Apart from impacting fitness under temperature stress, hopanoids are dispensable for vegetative cells under other stress conditions. However, hopanoids are required for stress tolerance in akinetes, a resting survival cell type. While 2‐methylated hopanoids do not appear to contribute to any stress phenotype, total hopanoids and to a lesser extent 2‐methylhopanoids were found to promote the formation of cyanophycin granules in akinetes. Finally, although hopanoids support symbiotic interactions between Alphaproteobacteria and plants, they do not appear to facilitate symbiosis between N. punctiforme and the hornwort Anthoceros punctatus. Collectively, these findings support interpreting hopanes as general environmental stress biomarkers. If hopanoid‐mediated enhancement of nitrogen‐rich storage products turns out to be a conserved phenomenon in other organisms, a better understanding of this relationship may help us parse the enrichment of 2‐methylhopanes in the rock record during episodes of disrupted nutrient cycling.     

Varietal similarities and differences in the polycyclic isopentenoid composition of sorghum

The intraspecific similarities and differences among the various polycyclic isopentenoids (sterols and pentacyclic triterpenes) which occur in leaf tissue and surface wax from three varieties of flowering Sorghum bicolor; G499 GBR, BOK 8 and IS 809, have been determined. The three varieties exhibited differences in phenotypic characters (e.g. shoot height) and pest resistance. While sterol and pentacyclic amyroid compositions were similar in the three varieties, significant differences were evident in the qualitative distribution of the migrated hopanoids. One variety, IS 809, which is phenotypically short and resistant, contained a single migrated hopanoid, sorghumol Alternatively, the other two varieties, both phenotypically tall and one-G 499 GBR-resistant, the other-BOK 8-susceptible, contained a mixture of four Δ⁹⁽¹¹⁾-migrated hopanoids, i.e. sorghumol and three of its stereoisomers, and the Δ⁵⁽⁶⁾-migrated hopanoid simiarenol. While ketones corresponding to the Δ⁹⁽¹¹⁾-migrated hopanoids were detected in the three varieties, the ketone of the Δ⁵⁽⁶⁾-migrated hopanoid was apparently absent. In contrast to other graminaceous plants, the predominant C-3 derivatives did not include the C-3 methyl ethers, but did include esters and polar conjugates. Significant differences apparent in amounts of pentacyclic triterpenes were not apparent in the total amount of sterols extracted from the leaves (including surface wax) of the three varieties. Since S. bicolor varietal differences occurred only among the migrated hopanoids (found in mature leaves) it would appear that pentacyclic triterpenes, unlike sterols, have greater applicability as chemotaxonomic indices for intraspecific relationships in sorghum.     

Targeted genomic detection of biosynthetic pathways: anaerobic production of hopanoid biomarkers by a common sedimentary microbe

The lipid biomarker principle requires that preservable molecules (molecular fossils) carry specific taxonomic, metabolic, or environmental information. Historically, an empirical approach was used to link specific taxa with the compounds they produce. The lipids extracted from numerous, but randomly cultured species provided the basis for the interpretation of biomarkers in both modern environments and in the geological record. Now, with the rapid sequencing of hundreds of microbial genomes, a more focused genomic approach can be taken to test phylogenetic patterns and hypotheses about the origins of biomarkers. Candidate organisms can be selected for study on the basis of genes that encode proteins fundamental to the synthesis of biomarker compounds. Hopanoids, a class of pentacyclic triterpenoid lipid biomarkers, provide an illustrative example. For many years, interpretations of biomarker data were made with the assumption that hopanoids are produced only by aerobic organisms. However, the recent discovery of ¹³C‐depleted hopanoids in environments undergoing anaerobic methane oxidation and in enrichment cultures of anammox planctomycetes indicates that some hopanoids are produced anaerobically. To further examine the potential distribution of hopanoid biosynthesis by anaerobes, we searched publicly available genomic databases for the presence of squalene‐hopene cyclase genes in known obligate or facultative anaerobes. Here we present evidence that Geobacter sulfurreducens, Geobacter metallireducens, and Magnetospirillum magnetotacticum, all bacteria common in anoxic environments, have the appropriate genes for hopanoid biosynthesis. We further show that these data accurately predict that G. sulfurreducens does produce a variety of complex hopanoids under strictly anaerobic conditions in pure culture.     

Hopanoid lipids in Bradyrhizobium and other plant-associated bacteria and cloning of the Bradyrhizobium japonicum squalene-hopene cyclase gene

Hopanoid lipids have been discovered recently in a number of nitrogen-fixing soil bacteria and in Bradyrhizobium bacteria which fix nitrogen in association with legume plants. We report here an investigation of the hopanoid content in an additional number of soil bacteria capable of living in close association with plants. Of the strains investigated, hopanoids were discovered in phototrophic, nitrogen-fixing bacteria and in an extended number of Bradyrhizobium strains. Strains in which hopanoids so far have not been found belong to the following genera: Rhizobium, Sinorhizobium, Phyllobacterium, Agrobacterium, and Azoarcus. To address the function of hopanoids in Bradyrhizobium, we cloned the gene coding for a key enzyme of hopanoid biosynthesis, the squalene-hopene cyclase, and expressed the gene in E. coli. The recombinant enzyme catalyzed in vitro the cyclization of squalene to hopanoid derivatives.Abbreviations SHC squalene-hopene cyclase - shc squalene-hopene cyclase gene     

Oxidation of Fe(II) leads to increased C‐2 methylation of pentacyclic triterpenoids in the anoxygenic phototrophic bacterium Rhodopseudomonas palustris strain TIE‐1

Hopanoids are among the most widespread biomarkers of bacteria that are used as indicators for past and present bacterial activity. Our understanding of the production, function, and distribution of hopanoids in bacteria has improved greatly, partly due to genetic, culture‐independent studies. Culture‐based studies are important to determine hopanoid function and the environmental conditions under which these compounds are produced. This study compares the lipid inventory of Rhodopseudomonas palustris strain TIE‐1 under anoxic photoautotrophic conditions using either H₂ or Fe(II) as electron donor. The high amount to which adenosylhopane is produced irrespective of the used electron donor suggests a specific function of this compound rather than its exclusive role as an intermediate in bacteriohopanepolyol biosynthesis. C‐2 methylated hopanoids and tetrahymanol account for as much as 59% of the respective C‐2 methylated/non‐methylated homologs during growth with Fe(II) as electron donor, as compared with 24% C‐2 methylation for growth with H₂. This observation reveals that C‐2 methylated hopanoids have a specific function and are preferentially synthesized in response to elevated Fe(II) concentrations. The presence of C‐2 methylated pentacyclic triterpenoids has commonly been used as a biosignature for the interpretation of paleoenvironments. These new findings suggest that increased C‐2 methylation may indicate anoxic ferrous conditions, in addition to other environmental stressors that have been previously reported.     

A general protein O‐glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O‐linked protein glycosylation system in B. cenocepacia K56‐2. The PglL_Bc O‐oligosaccharyltransferase (O‐OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N‐glycosylation system to a Neisseria meningitides‐derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56‐2, PglL_Bc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc‐HexNAc‐Hex, which is unrelated to the O‐antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post‐translational modification in Bcc with implications for pathogenesis.     

Burkholderia cenocepacia J2315 escapes to the cytosol and actively subverts autophagy in human macrophages

Selective autophagy functions to specifically degrade cellular cargo tagged by ubiquitination, including bacteria. Strains of the Burkholderia cepacia complex (Bcc) are opportunistic pathogens that cause life‐threatening infections in patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD). While there is evidence that defective macrophage autophagy in a mouse model of CF can influence B. cenocepacia susceptibility, there have been no comprehensive studies on how this bacterium is sensed and targeted by the host autophagy response in human macrophages. Here, we describe the intracellular life cycle of B. cenocepacia J2315 and its interaction with the autophagy pathway in human cells. Electron and confocal microscopy analyses demonstrate that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers theselective autophagy pathway, and the recruitment of ubiquitin, the ubiquitin‐binding adaptors p62 and NDP52 and the autophagosome membrane‐associated protein LC3B, to the bacterial vicinity. However, despite recruitment of these key autophagy pathway effectors, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. We find that a pre‐infection increase in cellular autophagy flux can significantly inhibit B. cenocepacia replication and that lower autophagy flux in macrophages from immunocompromised CGD patients could contribute to increased B. cenocepacia susceptibility, identifying autophagy manipulation as a potential therapeutic approach to reduce bacterial burden in B. cenocepacia infections.     

Content and composition of hopanoids in Zymomonas mobilis under various growth conditions.

By using a new method for quantification of the different hopanoid derivatives, a total hopanoid content of about 30 mg/g (dry cell weight) was observed in Zymomonas mobilis. This value is the highest reported for bacteria so far. The major hopanoids in Z. mobilis were the ether and glycosidic derivatives of tetrahydroxy-bacteriohopane, constituting about 41 and 49% of the total hopanoids. Tetrahydroxybacteriohopane itself, diplopterol, and hopene made up about 6, 3, and 1%, respectively. Only minor changes in hopanoid composition were observed with changes in growth conditions. Earlier reports on a correlation between hopanoid content and ethanol concentration in the medium could not be confirmed. Over a wide range of ethanol concentrations (5 to 60 g/liter), growth rates (0.08 to 0.25 h-1), and temperatures (25 to 37 degrees C), the molar ratio of hopanoids to phospholipids in the cells amounted to about 0.7. Only at growth rates of greater than 0.30 h-1 did the molar ratio increase to about 1.     

Draft Genome Sequence of Rhodomicrobium udaipurense JA643T with Special Reference to Hopanoid Biosynthesis

Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643^T isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100^T and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group containing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643^T will be useful in understanding the functional features of hopanoids in this bacterium.