Hopanoid Biosynthesis Research Articles

Integrative Genomics Sheds Light on Evolutionary Forces Shaping the Acidithiobacillia Class Acidophilic Lifestyle.

Extreme acidophiles thrive in environments rich in protons (pH values <3) and often high levels of dissolved heavy metals. They are distributed across the three domains of the Tree of Life including members of the Proteobacteria. The Acidithiobacillia class is formed by the neutrophilic genus Thermithiobacillus along with the extremely acidophilic genera Fervidacidithiobacillus, Igneacidithiobacillus, Ambacidithiobacillus, and Acidithiobacillus. Phylogenomic reconstruction revealed a division in the Acidithiobacillia class correlating with the different pH optima that suggested that the acidophilic genera evolved from an ancestral neutrophile within the Acidithiobacillia. Genes and mechanisms denominated as “first line of defense” were key to explaining the Acidithiobacillia acidophilic lifestyle including preventing proton influx that allows the cell to maintain a near-neutral cytoplasmic pH and differ from the neutrophilic Acidithiobacillia ancestors that lacked these systems. Additional differences between the neutrophilic and acidophilic Acidithiobacillia included the higher number of gene copies in the acidophilic genera coding for “second line of defense” systems that neutralize and/or expel protons from cell. Gain of genes such as hopanoid biosynthesis involved in membrane stabilization at low pH and the functional redundancy for generating an internal positive membrane potential revealed the transition from neutrophilic properties to a new acidophilic lifestyle by shaping the Acidithiobacillaceae genomic structure. The presence of a pool of accessory genes with functional redundancy provides the opportunity to “hedge bet” in rapidly changing acidic environments. Although a core of mechanisms for acid resistance was inherited vertically from an inferred neutrophilic ancestor, the majority of mechanisms, especially those potentially involved in resistance to extremely low pH, were obtained from other extreme acidophiles by horizontal gene transfer (HGT) events.

Marine and terrestrial nitrifying bacteria are sources of diverse bacteriohopanepolyols

Hopanoid lipids, bacteriohopanols and bacteriohopanepolyols, are membrane components exclusive to bacteria. Together with their diagenetic derivatives, they are commonly used as biomarkers for specific bacterial groups or biogeochemical processes in the geologic record. However, the sources of hopanoids to marine and freshwater environments remain inadequately constrained. Recent marker gene studies suggest a widespread potential for hopanoid biosynthesis in marine bacterioplankton, including nitrifying (i.e., ammonia- and nitrite-oxidizing) bacteria. To explore their hopanoid biosynthetic capacities, we studied the distribution of hopanoid biosynthetic genes in the genomes of cultivated and uncultivated ammonia-oxidizing (AOB), nitrite-oxidizing (NOB), and complete ammonia-oxidizing (comammox) bacteria, finding that biosynthesis of diverse hopanoids is common among seven of the nine presently cultivated clades of nitrifying bacteria. Hopanoid biosynthesis genes are also conserved among the diverse lineages of bacterial nitrifiers detected in environmental metagenomes. We selected seven representative NOB isolated from marine, freshwater, and engineered environments for phenotypic characterization. All tested NOB produced diverse types of hopanoids, with some NOB producing primarily diploptene and others producing primarily bacteriohopanepolyols. Relative and absolute abundances of hopanoids were distinct among the cultures and dependent on growth conditions, such as oxygen and nitrite limitation. Several novel nitrogen-containing bacteriohopanepolyols were tentatively identified, of which the so called BHP-743.6 was present in all NOB. Distinct carbon isotopic signatures of biomass, hopanoids, and fatty acids in four tested NOB suggest operation of the reverse tricarboxylic acid cycle in Nitrospira spp. and Nitrospina gracilis and of the Calvin-Benson-Bassham cycle for carbon fixation in Nitrobacter vulgaris and Nitrococcus mobilis. We suggest that the contribution of hopanoids by NOB to environmental samples could be estimated by their carbon isotopic compositions. The ubiquity of nitrifying bacteria in the ocean today and the antiquity of this metabolic process suggest the potential for significant contributions to the geologic record of hopanoids.

Genome analysis suggests the bacterial family Acetobacteraceae is a source of undiscovered specialized metabolites

Acetobacteraceae is an economically important family of bacteria that is used for industrial fermentation in the food/feed sector and for the preparation of sorbose and bacterial cellulose. It comprises two major groups: acetous species (acetic acid bacteria) associated with flowers, fruits and insects, and acidophilic species, a phylogenetically basal and physiologically heterogeneous group inhabiting acid or hot springs, sludge, sewage and freshwater environments. Despite the biotechnological importance of the family Acetobacteraceae, the literature does not provide any information about its ability to produce specialized metabolites. We therefore constructed a phylogenomic tree based on concatenated protein sequences from 141 type strains of the family and predicted the presence of small-molecule biosynthetic gene clusters (BGCs) using the antiSMASH tool. This dual approach allowed us to associate certain biosynthetic pathways with particular taxonomic groups. We found that acidophilic and acetous species contain on average ~ 6.3 and ~ 3.4 BGCs per genome, respectively. All the Acetobacteraceae strains encoded proteins involved in hopanoid biosynthesis, with many also featuring genes encoding type-1 and type-3 polyketide and non-ribosomal peptide synthases, and enzymes for aryl polyene, lactone and ribosomal peptide biosynthesis. Our in silico analysis indicated that the family Acetobacteraceae is a potential source of many undiscovered bacterial metabolites and deserves more detailed experimental exploration.

Hopanoid Hopene Locates in the Interior of Membranes and Affects Their Properties.

Hopanoids are proposed as sterol surrogates in some bacteria, and it has been proved that some hopanoids are able to induce a liquid-order phase state in lipid membranes. The members of this group of molecules have diverse structures, and not all of them have been studied in detail yet. Here, we study membranes with the hopanoid hopene (hop-22 (29)-ene or diploptene), which is the product of the cycling of squalene by squalene-hopene cyclase, and thus is present in the first step of hopanoid biosynthesis. Hopene is particularly interesting because it lacks a polar head group, which opens the question of how does this molecule accommodate in a lipid membrane, and what are the effects promoted by its presence. In order to get an insight into this, we prepared monolayers and bilayers of a phospholipid with hopene and studied their properties in comparison with pure phospholipid membranes, and with the sterol cholesterol or the hopanoid diplopterol. Film stiffness, shear viscosity, and bending dynamics were very affected by the presence of hopene, while zeta-potential, generalized polarization of Laurdan, and conductivity were affected moderately by this molecule. The results suggest that at very low percentages, hopene locates parallel to the phospholipid molecules, while the excess of the hopene molecules stays between leaflets, as previously proposed using molecular dynamics simulations.

Physiological, chemotaxonomic and genomic characterization of two novel piezotolerant bacteria of the family Marinifilaceae isolated from sulfidic waters of the Black Sea

Diversity analyses of microbial enrichments obtained from deep sulfidic water (2000 m) collected from the Black Sea indicated the presence of eleven novel putative lineages of bacteria affiliated to the family Marinifilaceae of the phylum Bacteroidetes. Pure cultures were obtained for four strains (i.e. M1PT, M3P, A4T and 44) of this family, which could be grouped into two different clades based on their 16S rRNA gene sequences. All four strains were Gram-negative, rod-shaped and facultative anaerobic bacteria. The genomes of all strains were sequenced and physiological analyses were performed. All strains utilized a wide range of carbon sources, which was supported by the presence of the pathways involved in carbon utilization encoded by their genomes. The strains were able to grow at elevated hydrostatic pressure (up to 50 MPa), which coincided with increased production of unsaturated and branched fatty acids, and a decrease in hydroxy fatty acids. Intact polar lipid analysis of all four strains showed the production of ornithine lipids, phosphatidylethanolamines and capnine lipids as major intact polar lipids (IPLs). Genes involved in hopanoid biosynthesis were also identified. However, bacteriohopanepolyols (BHPs) were not detected in the strains. Based on distinct physiological, chemotaxonomic, genotypic and phylogenetic differences compared to other members of the genera Ancylomarina and Labilibaculum, it was concluded that strains M1PT and A4T represented two novel species for which the names Ancylomarina euxinus sp. nov. and Labilibaculum euxinus sp. nov., respectively, are proposed.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Origin and Evolution of Polycyclic Triterpene Synthesis.

Polycyclic triterpenes are members of the terpene family produced by the cyclization of squalene. The most representative polycyclic triterpenes are hopanoids and sterols, the former are mostly found in bacteria, whereas the latter are largely limited to eukaryotes, albeit with a growing number of bacterial exceptions. Given their important role and omnipresence in most eukaryotes, contrasting with their scant representation in bacteria, sterol biosynthesis was long thought to be a eukaryotic innovation. Thus, their presence in some bacteria was deemed to be the result of lateral gene transfer from eukaryotes. Elucidating the origin and evolution of the polycyclic triterpene synthetic pathways is important to understand the role of these compounds in eukaryogenesis and their geobiological value as biomarkers in fossil records. Here, we have revisited the phylogenies of the main enzymes involved in triterpene synthesis, performing gene neighborhood analysis and phylogenetic profiling. Squalene can be biosynthesized by two different pathways containing the HpnCDE or Sqs proteins. Our results suggest that the HpnCDE enzymes are derived from carotenoid biosynthesis ones and that they assembled in an ancestral squalene pathway in bacteria, while remaining metabolically versatile. Conversely, the Sqs enzyme is prone to be involved in lateral gene transfer, and its emergence is possibly related to the specialization of squalene biosynthesis. The biosynthesis of hopanoids seems to be ancestral in the Bacteria domain. Moreover, no triterpene cyclases are found in Archaea, invoking a potential scenario in which eukaryotic genes for sterol biosynthesis assembled from ancestral bacterial contributions in early eukaryotic lineages.

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.

Extended Hopanoid Loss Reduces Bacterial Motility and Surface Attachment and Leads to Heterogeneity in Root Nodule Growth Kinetics in a Bradyrhizobium-Aeschynomene 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 hopanoid loss can be fully explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen-fixation levels. Using a single-nodule tracking approach to quantify 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 the 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.

Transcriptome analysis of hopanoid deficient mutant of Rhodopseuodomonas palustris TIE-1

All three domains of life have an ordered plasma membrane which is pivotal in the selective fitness of primitive life. Like cholesterol in eukaryotes, hopanoids are important in bacteria to modulate membrane order. Hopanoids are pentacyclic triterpenoid lipids biosynthesised in many eubacteria, few ferns and lichens. Hopanoid modulates outer membrane order and hopanoid deficiency results in the weakened structural integrity of the membrane which may in turn affect the other structures within or spanning the cell envelope and contributing to various membrane functions. Hence, to decipher the role of hopanoid, genome-wide transcriptome of wild-type and Δshc mutant of Rhodopseudomonas palustris TIE-1 was studied which indicated 299 genes were upregulated and 306 genes were downregulated in hopanoid deficient mutant, representing ∼11.5% of the genome. Thirty-eight genes involved in chemotaxis, response to stimuli and signal transduction were differentially regulated and impaired motility in hopanoid deficient mutant showed that hopanoid plays a crucial role in chemotaxis. The docking study demonstrated that diguanylate cyclase which catalyses the synthesis of secondary messenger exhibited the capability to interact with hopanoids and might be confederating in chemotaxis and signal transduction. Seventy-four genes involved in membrane transport were differentially expressed and cell assays also explicit that the multidrug transport is compromised in Δshc mutant. Membrane transport is reliant on hopanoids which may explain the basis for previous observations linking hopanoids to antibiotic resistance. Disturbing the membrane order by targeting lipid synthesis can be a possible novel approach in developing new antimicrobials and hopanoid biosynthesis could be a potential target.

Draft Genome Sequence of Methylovulum psychrotolerans Sph1 T , an Obligate Methanotroph from Low-Temperature Environments

ABSTRACTMethylovulum psychrotolerans Sph1T is an aerobic, obligate methanotroph, which was isolated from cold methane seeps in West Siberia. This bacterium possesses only a particulate methane monooxygenase and is widely distributed in low-temperature environments. Strain Sph1T has the genomic potential for biosynthesis of hopanoids required for the maintenance of intracytoplasmic membranes.

Hopanoid lipids: from membranes to plant-bacteria interactions.

Lipid research represents a frontier for microbiology, as showcased by hopanoid lipids. Hopanoids, which resemble sterols and are found in the membranes of diverse bacteria, have left an extensive molecular fossil record. They were first discovered by petroleum geologists. Today, hopanoid-producing bacteria remain abundant in various ecosystems, such as the rhizosphere. Recently, great progress has been made in our understanding of hopanoid biosynthesis, facilitated in part by technical advances in lipid identification and quantification. A variety of genetically tractable, hopanoid-producing bacteria have been cultured, and tools to manipulate hopanoid biosynthesis and detect hopanoids are improving. However, we still have much to learn regarding how hopanoid production is regulated, how hopanoids act biophysically and biochemically, and how their production affects bacterial interactions with other organisms, such as plants. The study of hopanoids thus offers rich opportunities for discovery.

Pheno- and Genotyping of Hopanoid Production in Acidobacteria.

Hopanoids are pentacyclic triterpenoid lipids synthesized by different bacterial groups. Methylated hopanoids were believed to be exclusively synthesized by cyanobacteria and aerobic methanotrophs until the genes encoding for the methylation at the C-2 and C-3 position (hpnP and hpnR) were found to be widespread in the bacterial domain, invalidating their use as specific biomarkers. These genes have been detected in the genome of the Acidobacterium “Ca. Koribacter versatilis,” but our knowledge of the synthesis of hopanoids and the presence of genes of their biosynthetic pathway in other member of the Acidobacteria is limited. We analyzed 38 different strains of seven Acidobacteria subdivisions (SDs 1, 3, 4, 6, 8, 10, and 23) for the presence of C30 hopenes and C30+ bacteriohopane polyols (BHPs) using the Rohmer reaction. BHPs and/or C30 hopenes were detected in all strains of SD1 and SD3 but not in SD4 (excepting Chloracidobacterium thermophilum), 6, 8, 10, and 23. This is in good agreement with the presence of genes required for hopanoid biosynthesis in the 31 available whole genomes of cultivated Acidobacteria. All genomes encode the enzymes involved in the non-mevalonate pathway ultimately leading to farnesyl diphosphate but only SD1 and 3 Acidobacteria and C. thermophilum encode all three enzymes required for the synthesis of squalene, its cyclization (shc), and addition and modification of the extended side chain (hpnG, hpnH, hpnI, hpnJ, hpnO). In almost all strains, only tetrafunctionalized BHPs were detected; three strains contained variable relative abundances (up to 45%) of pentafunctionalized BHPs. Only “Ca. K. versatilis” contained methylated hopanoids (i.e., 2,3-dimethyl bishomohopanol), although in low (<10%) amounts. These genes are not present in any other Acidobacterium, consistent with the absence of methylated BHPs in the other examined strains. These data are in agreement with the scattered occurrence of methylated BHPs in other bacterial phyla such as the Alpha-, Beta-, and Gammaproteobacteria and the Cyanobacteria, limiting their biomarker potential. Metagenomes of Acidobacteria were also examined for the presence of genes required for hopanoid biosynthesis. The complete pathway for BHP biosynthesis was evident in SD2 Acidobacteria and a group phylogenetically related to SD1 and SD3, in line with the limited occurrence of BHPs in acidobacterial cultures.

Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

Genomic approaches toward understanding the actinorhizal symbiosis: an update on the status of the Frankia genomes

The actinorhizal symbiosis is a mutualistic relationship between an actinobacterium from the genus Frankia and a wide variety of dicotyledonous plants representing 8 different families of angiosperms. Molecular phylogenetic approaches have identified four major Frankia lineages that have distinctive plant host ranges. Since the first published three Frankia genomes, an effort was undertaken to provide full genomic databases covering all four well established Frankia lineages and to provide depth of the number of strains covered. Here, we report on the updated status of these sequencing efforts. At present, there are 25 complete or draft Frankia genomes that have been sequenced and annotated, and several others are now in the pipeline being sequenced. An overview of the Frankia genomes will be presented focusing on their general genomic properties including size of the pan- and core-gene pool, size relationship and genome plasticity. Furthermore, a description of biosynthetic potential and a discussion about genes (nitrogenase, hopanoid biosynthesis, truncated hemoglobin, hydrogenase uptake gene clusters) involved in the symbiosis will be discussed. The absence of common nod genes within these Frankia genomes provides clues about the host-microbe recognition process for the actinorhizal symbiosis

Absolute Configuration of Hydroxysqualene. An Intermediate in Bacterial Hopanoid Biosynthesis.

Squalene (SQ) is a key intermediate in hopanoid biosynthesis. Many bacteria synthesize SQ from farnesyl diphosphate (FPP) in three steps: FPP to (1R,2R,3R)-presqualene diphosphate (PSPP), (1R,2R,3R)-PSPP to hydroxysqualene (HSQ), and HSQ to SQ. Chemical, biochemical, and spectroscopic methods were used to establish that HSQ synthase synthesizes (S)-HSQ. In contrast, eukaryotic squalene synthase catalyzes solvolysis of (1R,2R,3R)-PSPP to give (R)-HSQ. The bacterial enzyme that reduces HSQ to SQ does not accept (R)-HSQ as a substrate.

Salt Stress Induced Changes in the Exoproteome of the Halotolerant Bacterium Tistlia consotensis Deciphered by Proteogenomics

The ability of bacteria to adapt to external osmotic changes is fundamental for their survival. Halotolerant microorganisms, such as Tistlia consotensis, have to cope with continuous fluctuations in the salinity of their natural environments which require effective adaptation strategies against salt stress. Changes of extracellular protein profiles from Tistlia consotensis in conditions of low and high salinities were monitored by proteogenomics using a bacterial draft genome. At low salinity, we detected greater amounts of the HpnM protein which is involved in the biosynthesis of hopanoids. This may represent a novel, and previously unreported, strategy by halotolerant microorganisms to prevent the entry of water into the cell under conditions of low salinity. At high salinity, proteins associated with osmosensing, exclusion of Na+ and transport of compatible solutes, such as glycine betaine or proline are abundant. We also found that, probably in response to the high salt concentration, T. consotensis activated the synthesis of flagella and triggered a chemotactic response neither of which were observed at the salt concentration which is optimal for growth. Our study demonstrates that the exoproteome is an appropriate indicator of adaptive response of T. consotensis to changes in salinity because it allowed the identification of key proteins within its osmoadaptive mechanism that had not previously been detected in its cell proteome.

Biosynthesis of Squalene from Farnesyl Diphosphate in Bacteria: Three Steps Catalyzed by Three Enzymes.

Squalene (SQ) is an intermediate in the biosynthesis of sterols in eukaryotes and a few bacteria and of hopanoids in bacteria where they promote membrane stability and the formation of lipid rafts in their hosts. The genes for hopanoid biosynthesis are typically located on clusters that consist of four highly conserved genes—hpnC, hpnD, hpnE, and hpnF—for conversion of farnesyl diphosphate (FPP) to hopene or related pentacyclic metabolites. While hpnF is known to encode a squalene cyclase, the functions for hpnC, hpnD, and hpnE are not rigorously established. The hpnC, hpnD, and hpnE genes from Zymomonas mobilis and Rhodopseudomonas palustris were cloned into Escherichia coli, a bacterium that does not contain genes homologous to hpnC, hpnD, and hpnE, and their functions were established in vitro and in vivo. HpnD catalyzes formation of presqualene diphosphate (PSPP) from two molecules of FPP; HpnC converts PSPP to hydroxysqualene (HSQ); and HpnE, a member of the amine oxidoreductase family, reduces HSQ to SQ. Collectively the reactions catalyzed by these three enzymes constitute a new pathway for biosynthesis of SQ in bacteria.

Activation of Cryptic hop Genes from Streptomyces peucetius ATCC 27952 Involved in Hopanoid Biosynthesis

Genes encoding enzymes with sequence similarity to hopanoids biosynthetic enzymes of other organisms were cloned from the hopanoid (hop) gene cluster of Streptomyces peucetius ATCC 27952 and transformed into Streptomyces venezuelae YJ028. The cloned fragments contained four genes, all transcribed in one direction. These genes encode polypeptides that resemble polyprenyl diphosphate synthase (hopD), squalene-phytoene synthases (hopAB), and squalenehopene cyclase (hopE). These enzymes are sufficient for the formation of the pentacyclic triterpenoid lipid, hopene. The formation of hopene was verified by gas chromatography/ mass spectrometry.

Ribosylhopane, a novel bacterial hopanoid, as precursor of C35 bacteriohopanepolyols in Streptomyces coelicolor A3(2).

Wild-type Streptomyces coelicolor A3(2) produces aminobacteriohopanetriol as the only elongated C35 hopanoid. The hopanoid phenotype of two mutants bearing a deletion of genes from a previously identified hopanoid biosynthesis gene cluster provides clues to the formation of C35 bacteriohopanepolyols. orf14 encodes a putative nucleosidase; its deletion induces the accumulation of adenosylhopane as it cannot be converted into ribosylhopane. orf18 encodes a putative transaminase; its deletion results in the accumulation of adenosylhopane, ribosylhopane, and bacteriohopanetetrol. Ribosylhopane was postulated twenty years ago as a precursor for bacterial hopanoids but was never identified in a bacterium. Absence of the transaminase encoded by orf18 prevents the reductive amination of ribosylhopane into aminobacteriohopanetriol and induces its accumulation. Its reduction by an aldose-reductase-like enzyme produces bacteriohopanetetrol, which is normally not present in S. coelicolor.