Rachael M. Morgan-Kiss

A new Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic β-oxidation pathway

Escherichia coli uses fatty acids as a sole carbon and energy source during aerobic growth by means of the enzymes encoded by the fad regulon. We report that this bacterium can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor such as nitrate is available. This anaerobic utilization pathway is distinct from the well‐studied aerobic pathway in that (i) it proceeds normally in mutant strains lacking various enzymes of the aerobic pathway; (ii) it functions with fatty acids (octanoate and decanoate) that cannot be used by wild‐type E. coli strains under aerobic conditions; and (iii) super‐repressor mutants of the fadR regulatory locus that block aerobic growth on fatty acids fail to block the anaerobic pathway. We have identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty acids. A strong FadR binding site was detected upstream of the yfcY gene consistent with microarray analyses, indicating that yfcYX expression is negatively regulated by FadR under aerobic growth conditions. In contrast, transcriptional regulation of ydiD appears to be independent of FadR, and anaerobic growth on fatty acids is not under FadR control. These three genes are conserved in the available genome sequences of pathogenic E. coli , Shigella and Salmonella strains.

Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity

Expression systems based on the Escherichia coli arabinose operon PBAD promoter exhibit the all-or-nothing (autocatalytic) induction of expression that was first documented in the lac operon. Under conditions of subsaturating levels of inducer, some of the cells of the population are fully induced, whereas other cells remain uninduced. Recently, a new AraE transporter system was reported to have circumvented the problem of autocatalytic expression in the pBAD expression vectors and to provide graded and homogeneous cell-to-cell expression in the presence of variable inducer concentrations [Khlebnikov, A., Risa, O., Skaug, T., Carrier, T. A. & Keasling, J. D. (2000) J. Bacteriol. 182, 7029–7034]. However, we report that nonuniform gene expression in the AraE system was readily detectable by the use of mutant green fluorescent proteins that are rapidly degraded in E. coli. We report an approach to avoid all-or-nothing induction of the pBAD promoter; the use of a mutant LacY transporter in a strain deficient in both arabinose transport (araE araFGH) and degradation (araBAD). This mutant LacY protein performs facilitated diffusion of arabinose resulting in homogeneous expression of an unstable GFP that is maintained over extended incubation times at subsaturating levels of inducer. This approach is readily adapted to other sugar-regulated expression systems.

The Escherichia coli fadK (ydiD) gene encodes an anerobically regulated short chain acyl-CoA synthetase.

We recently reported a new metabolic competency for Escherichia coli, the ability to degrade and utilize fatty acids of various chain lengths as sole carbon and energy sources (Campbell, J. W., Morgan-Kiss, R. M., and Cronan J. E. (2003) Mol. Microbiol. 47, 793–805). This β-oxidation pathway is distinct from the previously described aerobic fatty acid degradation pathway and requires enzymes encoded by two operons, yfcYX and ydiQRSTD. The yfcYX operon (renamed fadIJ) encodes enzymes required for hydration, oxidation, and thiolytic cleavage of the acyl chain. The ydiQRSTD operon encodes a putative acyl-CoA synthetase, ydiD (renamed fadK), as well as putative electron transport chain components. We report that FadK is as an acyl-CoA synthetase that has a preference for short chain length fatty acid substrates (<10 C atoms). The enzymatic mechanism of FadK is similar to other acyl-CoA synthetases in that it forms an acyl-AMP intermediate prior to the formation of the final acyl-CoA product. Expression of FadK is repressed during aerobic growth and is maximally expressed under anaerobic conditions in the presence of the terminal electron acceptor, fumarate.

Differential thermal effects on the energy distribution between photosystem II and photosystem I in thylakoid membranes of a psychrophilic and a mesophilic alga.

Sensitivity of the photosynthetic thylakoid membranes to thermal stress was investigated in the psychrophilic Antarctic alga Chlamydomonas subcaudata. C. subcaudata thylakoids exhibited an elevated heat sensitivity as indicated by a temperature-induced rise in F(o) fluorescence in comparison with the mesophilic species, Chlamydomonas reinhardtii. This was accompanied by a loss of structural stability of the photosystem (PS) II core complex and functional changes at the level of PSI in C. reinhardtii, but not in C. subcaudata. Lastly, C. subcaudata exhibited an increase in unsaturated fatty acid content of membrane lipids in combination with unique fatty acid species. The relationship between lipid unsaturation and the functioning of the photosynthetic apparatus under elevated temperatures is discussed.

Functional Analysis of Three Sulfide:Quinone Oxidoreductase Homologs in Chlorobaculum tepidum

Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo- and phototrophic growth in bacteria. The green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum) can grow on sulfide as the sole electron donor and sulfur source. C. tepidum contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in C. tepidum decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with >or=6 mM sulfide. Mutation of both CT0117 and CT1087 in C. tepidum completely abolished SQR activity, and the double mutant failed to grow with >or=4 mM sulfide. A C-terminal His(6)-tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in C. tepidum. CT0876 may support the growth of C. tepidum at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.

Protist diversity in a permanently ice-covered Antarctic Lake during the polar night transition

The McMurdo Dry Valleys of Antarctica harbor numerous permanently ice-covered lakes, which provide a year-round oasis for microbial life. Microbial eukaryotes in these lakes occupy a variety of trophic levels within the simple aquatic food web ranging from primary producers to tertiary predators. Here, we report the first molecular study to describe the vertical distribution of the eukaryotic community residing in the photic zone of the east lobe (ELB) and west lobe (WLB) of the chemically stratified Lake Bonney. The 18S ribosomal RNA (rRNA) libraries revealed vertically stratified populations dominated by photosynthetic protists, with a cryptophyte dominating shallow populations (ELB–6 m; WLB–10 m), a haptophyte occupying mid-depths (both lobes 13 m) and chlorophytes residing in the deepest layers (ELB–18 and 20 m; WLB–15 and 20 m) of the photic zone. A previously undetected stramenopile occurred throughout the water column of both lobes. Temporal variation in the eukaryotic populations was examined during the transition from Antarctic summer (24-h sunlight) to polar night (complete dark). Protist diversity was similar between the two lobes of Lake Bonney due to exchange between the photic zones of the two basins via a narrow bedrock sill. However, vertical and temporal variation in protist distribution occurred, indicating the influence of the unique water chemistry on the biology of the two dry valley watersheds.

The Lactococcus lactis FabF Fatty Acid Synthetic Enzyme Can Functionally Replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. colifabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.

The Antarctic psychrophile, Chlamydomonas raudensis Ettl (UWO241) (Chlorophyceae, Chlorophyta), exhibits a limited capacity to photoacclimate to red light

The psychrophilic Antarctic alga, Chlamydomonas raudensis Ettl (UWO241), grows under an extreme environment of low temperature and low irradiance of a limited spectral quality (blue‐green). We investigated the ability of C. raudensis to acclimate to long‐term imbalances in excitation caused by light quality through adjustments in photosystem stoichiometry. Log‐phase cultures of C. raudensis and C. reinhardtii grown under white light were shifted to either blue or red light for 12 h. Previously, we reported that C. raudensis lacks the ability to redistribute light energy via the short‐term mechanism of state transitions. However, similar to the model of mesophilic alga, C. reinhardtii, the psychrophile retained the capacity for long‐term adjustment in energy distribution between PSI and PSII by modulating the levels of PSI reaction center polypeptides, PsaA/PsaB, with minimal changes in the content of the PSII polypeptide, D1, in response to changes in light quality. The functional consequences of the modulation in PSI/PSII stoichiometry in the psychrophile were distinct from those observed in C. reinhardtii. Exposure of C. raudensis to red light caused 1) an inhibition of growth and photosynthetic rates, 2) an increased reduction state of the intersystem plastoquinone pool with concomitant increases in nonphotochemical quenching, 3) an uncoupling of the major light‐harvesting complex from the PSII core, and 4) differential thylakoid protein phosphorylation profiles compared with C. reinhardtii. We conclude that the characteristic low levels of PSI relative to PSII set the limit in the capacity of C. raudensis to photoacclimate to an environment enriched in red light.

Expression of Vibrio harveyi Acyl-ACP Synthetase Allows Efficient Entry of Exogenous Fatty Acids into the Escherichia coli Fatty Acid and Lipid A Synthetic Pathways

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Diversity and expression of RubisCO genes in a perennially ice-covered Antarctic lake during the polar night transition

ABSTRACT The autotrophic communities in the lakes of the McMurdo Dry Valleys, Antarctica, have generated interest since the early 1960s owing to low light transmission through the permanent ice covers, a strongly bimodal seasonal light cycle, constant cold water temperatures, and geographical isolation. Previous work has shown that autotrophic carbon fixation in these lakes provides an important source of organic matter to this polar desert. Lake Bonney has two lobes separated by a shallow sill and is one of several chemically stratified lakes in the dry valleys that support year-round biological activity. As part of an International Polar Year initiative, we monitored the diversity and abundance of major isoforms of RubisCO in Lake Bonney by using a combined sequencing and quantitative PCR approach during the transition from summer to polar winter. Form ID RubisCO genes related to a stramenopile, a haptophyte, and a cryptophyte were identified, while primers specific for form IA/B RubisCO detected a diverse autotrophic community of chlorophytes, cyanobacteria, and chemoautotrophic proteobacteria. Form ID RubisCO dominated phytoplankton communities in both lobes of the lake and closely matched depth profiles for photosynthesis and chlorophyll. Our results indicate a coupling between light availability, photosynthesis, and rbcL mRNA levels in deep phytoplankton populations. Regulatory control of rbcL in phytoplankton living in nutrient-deprived shallow depths does not appear to be solely light dependent. The distinct water chemistries of the east and west lobes have resulted in depth- and lobe-dependent variability in RubisCO diversity, which plays a role in transcriptional activity of the key gene responsible for carbon fixation.