Evaluating Metabolic Pathways and Biofilm Formation in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia has recently arisen as a prominent nosocomial pathogen because of its high antimicrobial resistance and ability to cause chronic respiratory infections. Often the infections are worsened by biofilm formation which enhances antibiotic tolerance. We have previously found that mutation of the gpmA gene, encoding the glycolytic enzyme phosphoglycerate mutase, impacts the formation of this biofilm on biotic and abiotic surfaces at early time points. This finding, indicating an association between carbon source and biofilm formation, led us to hypothesize that metabolism would influence S. maltophilia biofilm formation and planktonic growth. In the present study, we tested the impact of various growth substrates on biofilm levels and growth kinetics to determine metabolic requirements for these processes. We found that S. maltophilia wild type preferred amino acids versus glucose for planktonic and biofilm growth and that gpmA deletion inhibited growth in amino acids. Furthermore, supplementation of the ΔgpmA strain by glucose or ribose phenotypically complemented growth defects. These results suggest that S. maltophilia shuttles amino acid carbon through gluconeogenesis to an undefined metabolic pathway supporting planktonic and biofilm growth. Further evaluation of these metabolic pathways might reveal novel metabolic activities of this pathogen. IMPORTANCE Stenotrophomonas maltophilia is a prominent opportunistic pathogen that often forms biofilms during infection. However, the molecular mechanisms of virulence and biofilm formation are poorly understood. The glycolytic enzyme phosphoglycerate mutase appears to play a role in biofilm formation, and we used a mutant in its gene (gpmA) to probe the metabolic circuitry potentially involved in biofilm development. The results of our study indicate that S. maltophilia displays unique metabolic activities, which could be exploited for inhibiting growth and biofilm formation of this pathogen.

Pyridoxal and α-Ketoglutarate Independently Improve Function of Saccharomyces cerevisiae Thi5 in the Metabolic Network of Salmonella enterica.

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica. Function of Thi5 from Saccharomyces cerevisiae (ScThi5) required modification of the underlying metabolic network, while LpThi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of ScThi5 and report strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and ScThi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved ScThi5 function in S. enterica. Finally, directly increasing αKG and PLP with supplementation improved function of both ScThi5 and relevant variants of Thi5 from Legionella pneumophila (LpThi5). The data herein suggest structural differences between ScThi5 and LpThi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica. IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between ScThi5 and LpThi5 contributes to their functional differences in a Salmonella enterica host.

Analysis of the Manganese and MntR Regulon in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) is likely critical for host colonization. MntR is an Mn-dependent transcriptional regulator in C. diphtheriae that represses the expression of the mntABCD genes, which encode a putative ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between the wild type and an mntR deletion mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce various target genes in an Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts with the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and cadmium that could be alleviated by the additional deletion of the mntABCD transport locus, providing evidence that the MntABCD transporter functions as an Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen. IMPORTANCE Mechanisms for metal scavenging are critical to the survival and success of bacterial pathogens, including Corynebacterium diphtheriae. Metal import systems in pathogenic bacteria have been studied as possible vaccine components due to high conservation, critical functionality, and surface localization. In this study, we expand our understanding of the genes controlled by the global manganese regulator, MntR. We determined a role for the MntABCD transporter in manganese import using evidence from manganese and cadmium toxicity assays. Understanding the nutritional requirements of C. diphtheriae and the tools used to acquire essential metals will aid in the development of future vaccines.

Combinatorial Sensor Design in Caulobacter crescentus for Selective Environmental Uranium Detection.

The ability to detect uranium (U) through environmental monitoring is of critical importance for informing water resource protection and nonproliferation efforts. While technologies exist for environmental U detection, wide-area environmental monitoring, i.e. sampling coverage over large areas not known to possess U contamination, remains a challenging prospect that necessitates the development of novel detection approaches. Herein, we describe the development of a whole-cell U sensor by integrating two functionally independent, native U-responsive two-component signaling systems (TCS), UzcRS and UrpRS, within an AND gate circuit in the bacterium Caulobacter crescentus. Through leverage of the distinct but imperfect selectivity profiles of both TCS, this combinatorial approach enabled greater selectivity relative to a prior biosensor developed with UzcRS alone; no cross-reactivity was observed with most common environmental metals (e.g, Fe, As, Cu, Ca, Mg, Cd, Cr, Al) or the U decay-chain product Th, and the selectivity against Zn and Pb was significantly improved. In addition, integration of the UzcRS signal amplifier protein UzcY within the AND gate circuit further enhanced overall sensitivity and selectivity for U. The functionality of the sensor in an environmental context was confirmed by detection of U concentrations as low as 1 µM in groundwater samples. The results highlight the value of a combinatorial approach for constructing whole-cell sensors for the selective detection of analytes for which there are no known evolved regulators.

Positive and negative regulation of transferred nif genes mediated by indigenous GlnR in Gram-positive Paenibacillus polymyxa.

Ammonia is a major signal that regulates nitrogen fixation in most diazotrophs. Regulation of nitrogen fixation by ammonia in the Gram-negative diazotrophs is well-characterized. In these bacteria, this regulation occurs mainly at the level of nif (nitrogen fixation) gene transcription, which requires a nif-specific activator, NifA. Although Gram-positive and diazotrophic Paenibacilli have been extensively used as a bacterial fertilizer in agriculture, how nitrogen fixation is regulated in response to nitrogen availability in these bacteria remains unclear. An indigenous GlnR and GlnR/TnrA-binding sites in the promoter region of the nif cluster are conserved in these strains, indicating the role of GlnR as a regulator of nitrogen fixation. In this study, we for the first time reveal that GlnR of Paenibacillus polymyxa WLY78 is essentially required for nif gene transcription under nitrogen limitation, whereas both GlnR and glutamine synthetase (GS) encoded by glnA within glnRA operon are required for repressing nif expression under excess nitrogen. Dimerization of GlnR is necessary for binding of GlnR to DNA. GlnR in P. polymyxa WLY78 exists in a mixture of dimers and monomers. The C-terminal region of GlnR monomer is an autoinhibitory domain that prevents GlnR from binding DNA. Two GlnR-biding sites flank the -35/-10 regions of the nif promoter of the nif operon (nifBHDKENXhesAnifV). The GlnR-binding site Ⅰ (located upstream of -35/-10 regions of the nif promoter) is specially required for activating nif transcription, while GlnR-binding siteⅡ (located downstream of -35/-10 regions of the nif promoter) is for repressing nif expression. Under nitrogen limitation, GlnR dimer binds to GlnR-binding siteⅠ in a weak and transient association way and then activates nif transcription. During excess nitrogen, glutamine binds to and feedback inhibits GS by forming the complex FBI-GS. The FBI-GS interacts with the C-terminal domain of GlnR and stabilizes the binding affinity of GlnR to GlnR-binding site Ⅱ and thus represses nif transcription.

Identification of Differentially Regulated Edwardsiella ictaluri Proteins During Catfish Serum Treatment.

Edwardsiella ictaluri is a facultative, intracellular, gram-negative bacterium that causes enteric septicemia of catfish (ESC). Edwardsiella ictaluri is known to be resistant to defense mechanisms present in catfish serum, which might aid in its use of a host's bloodstream to become septicemic. However, the precise mechanisms of the survival of E. ictaluri in host serum are not known. Analysis of the response of E. ictaluri to the host serum treatment at a proteomic level might aid in the elucidation of its adaptation mechanisms against defense mechanisms present in catfish serum. Thus, the objective of this study was to identify differentially regulated proteins of E. ictaluri upon exposure to naïve catfish serum. Two-dimensional difference gel electrophoresis (2D-DIGE) followed by in-gel trypsin digestion and MALDI-TOF/TOF analysis were used for identification of differentially expressed E. ictaluri proteins. A total of 19 differentially regulated proteins (7 up- and 12 downregulated) were identified. Among those were four putative immunogenic proteins, two chaperones and eight proteins involved in the translational process, two nucleic acid degradation and integration proteins, two intermediary metabolism proteins, and one iron-ion-binding protein. Further research focusing on the functions of these differentially expressed proteins may reveal their roles in host adaptation by E. ictaluri.

Exometabolomic Profiling of Bacterial Strains as Cultivated Using Arabidopsis Root Extract as the Sole Carbon Source.

The ability of microorganisms to use root-derived metabolites as growth substrates is a key trait for success in the rhizospheric niche. However, few studies describe which specific metabolites are consumed or to what degree microbial strains differ in their substrate consumption patterns. Here, we present a liquid chromatography-mass spectrometry (MS) exometabolomic study of three bacterial strains cultivated using either glucose or Arabidopsis thaliana root extract as the sole carbon source. Two of the strains were previously isolated from field-grown Arabidopsis roots, the other is Escherichia coli, included as a comparison. When cultivated on root extract, a set of 62 MS features were commonly taken up by all three strains, with m/z values matching components of central metabolism (including amino acids and purine or pyrimidine derivatives). Escherichia coli took up very few MS features outside this commonly consumed set, whereas the root-inhabiting strains took up a much larger number of MS features, many with m/z values matching plant-specific metabolites. These measurements define the metabolic niche that each strain potentially occupies in the rhizosphere. Furthermore, we document many MS features released by these strains that could play roles in cross-feeding, antibiosis, or signaling. We present our methodological approach as a foundation for future studies of rhizosphere exometabolomics.

Characterization of the Serralysin-Like Gene of 'Candidatus Liberibacter solanacearum' Associated with Potato Zebra Chip Disease.

The nonculturable bacterium 'Candidatus Liberibacter solanacearum' is the causative agent of zebra chip disease in potato. Computational analysis of the 'Ca. L. solanacearum' genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The 'Ca. L. solanacearum' serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional 'Ca. L. solanacearum' serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the 'Ca. L. solanacearum' serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the 'Ca. L. solanacearum' serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-'Ca. L. solanacearum' serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the 'Ca. L. solanacearum' protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the 'Ca. L. solanacearum' serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.

VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae.

When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during 'uranium-shock' implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Effect of Zn2+ on halohydrin dehalogenase expression and accumulation through multi-parameter correlation research with Escherichia coli P84A/MC1061.

Using 5 Zn2+ supplementation strategies in a 50 L batch bioreactor named FUS-50L(A), possible correlations among Zn2+ content and addition timing, physiologic activity (PA), halohydrin dehalogenase (HheC) accumulation of Escherichia coli P84A/MC1061 were systematically investigated. First, Zn2+ was confirmed as the significant factor, and its optimal concentration for HheC expression was 3.87 mg/L through fermentation experiments in shaking flasks. Second, based on experimental results from the different strategies, it was found that PA, nutrient consumption rate (NCR) and specific growth rate (µ) for E. coli P84A/MC1061 were promoted in the log phase (4-8 h) under appropriate Zn2+ concentrations in the lag phase and late log phase. Furthermore cell biomass was also increased to a higher level and the maximum HheC activity (i.e. HheCmax) was increased by 9.80%, and the time to reach HheCmax was reduced from 16 to 12 hours. Furthermore, appropriate supplementation of Zn2+ caused higher µ for E. coli P84A/MC1061, which resulted in more rapid accumulation of increased acetic acid concentrations, leading to higher acetic acid consumption avoiding any negative effects on producing HheC because of carbon source being exhausted prematurely and acetic acid being consumed rapidly.

An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli.

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase ß subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na+ /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc.

Impact of phosphate and other medium components on physiological regulation of bacterial laccase production.

Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10-500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2-1%), with R. tangshanensis as an exception, or yeast extract (1.5-2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:541-548, 2017.

Microcystin Biosynthesis and mcyA Expression in Geographically Distinct Microcystis Strains under Different Nitrogen, Phosphorus, and Boron Regimes.

Roles of nutrients and other environmental variables in development of cyanobacterial bloom and its toxicity are complex and not well understood. We have monitored the photoautotrophic growth, total microcystin concentration, and microcystins synthetase gene (mcyA) expression in lab-grown strains of Microcystis NIES 843 (reference strain), KW (Wangsong Reservoir, South Korea), and Durgakund (Varanasi, India) under different nutrient regimes (nitrogen, phosphorus, and boron). Higher level of nitrogen and boron resulted in increased growth (avg. 5 and 6.5 Chl a mg/L, resp.), total microcystin concentrations (avg. 1.185 and 7.153 mg/L, resp.), and mcyA transcript but its expression was not directly correlated with total microcystin concentrations in the target strains. Interestingly, Durgakund strain had much lower microcystin content and lacked microcystin-YR variant over NIES 843 and KW. It is inferred that microcystin concentration and its variants are strain specific. We have also examined the heterotrophic bacteria associated with cyanobacterial bloom in Durgakund Pond and Wangsong Reservoir which were found to be enriched in Alpha-, Beta-, and Gammaproteobacteria and that could influence the bloom dynamics.

Iron-regulated gene ireA in avian pathogenic Escherichia coli participates in adhesion and stress-resistance.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis, which results in economic and welfare costs in the poultry industry worldwide. The pathogenesis of avian pathogenic E. coli strains is not well defined. Here, the function of an outer membrane protein encoded by the ireA gene of avian pathogenic E. coli strain DE205B was investigated. RESULTS: The ireA gene was distributed in 32.9 % (46/140) of tested E. coli strains, with high percentages in the phylogenetic ECOR groups B2 (58.8 %, 10/17) and D (55.9 %, 19/34). The gene expression level of ireA of APEC strain DE205B in high Fe M9 media was 1.8 times higher (P < 0.05) than that in low Fe M9 media. An ireA deletion mutant and complementary strain were constructed. Compared with the wild-type strain DE205B, the expression of most ferric uptake genes in the ireA deletion mutant were significantly upregulated (P < 0.05). The adhesion ability of the ireA deletion mutant to DF-1 cells was significantly decreased. The survival rate of ireA deletion mutant was reduced 21.17 % (P < 0.01), 25.42 (P < 0.05) and 70.0 % (P < 0.01) under alkali, high osmolarity, and low temperature (4 °C) conditions, respectively, compared with the wild-type strain. CONCLUSIONS: The results suggested that the protein encoded by the iron-regulated gene ireA has roles in adhesion and stress resistance in avian pathogenic E. coli.

TmcN is involved in ATP regulation of tautomycetin biosynthesis in Streptomyces griseochromogenes.

The regulatory mechanism of tautomycetin (TMC) biosynthesis remains largely unknown, although it has been of great interest to the pharmaceutical industry. Our previous study showed that intracellular adenosine triphosphate (inATP) level is negatively correlated with secondary metabolite biosynthesis in various Streptomyces spp. In this study, by exogenous treatment of ATP, we also found a negative correlation between TMC biosynthesis and inATP level in Streptomyces griseochromogenes (S. griseochromogenes). However, the underlying mechanism remains unclear. TmcN, a pathway-specific transcriptional regulator of TMC biosynthetic genes, was previously revealed as a large ATP-binding LuxR (LAL) family protein. The predicted amino acid sequence of TmcN shows highly conserved Walker A and B binding motifs, which suggest an ATPase function of TmcN. We therefore hypothesized that the ATPase domain of TmcN may play a role in sensing endogenous pool of ATP, and is thus involved in the ATP regulation of TMC biosynthesis. To test the hypothesis, we first explored the key residue that affects the ATPase activity of TmcN by amino acid sequence alignment and structural simulation. After that, we disrupted tmcN gene in S. griseochromogenes, and the tmcN or site-direct-mutated tmcN were re-introduced to get the complementary and ATPase domain disrupted strains. The transcription level of tmcN, TMC yield, and inATP, as well as the effect of ATP on TMC production of different mutants were evaluated. Deletion of tmcN or site-direct mutation of ATPase domain of TmcN in S. griseochromogenes significantly reduced the TMC production, and it was not affected by exogenous ATP treatment. In addition, a relatively high level of inATP was detected in tmcN deletion and site-direct mutation strains. Our results here suggested that TmcN, especially its ATPase domain, is involved in consuming of endogenous ATP pool and thus plays pivotal role in connecting the primary and secondary metabolite in S. griseochromogenes.

The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans.

UNLABELLED: The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism. IMPORTANCE: The identification of the gene product SMUL_1544 as a putative l-Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide-containing enzymes (e.g., reductive dehalogenases) showed specificity for their cobamide cofactors. It has recently been proposed that the structure of the linker moiety of norpseudo-B12 and the mode of binding of the EA-P linker to the PceA enzyme reflect the high specificity of the enzyme for its cofactor. Data reported herein do not support this idea. In fact, norpseudo-B12 was functional in the cobamide-dependent methionine biosynthesis of S. enterica, raising questions about the role of norcobamides in nature.

Further Insight into Polycyclization Cascades of Acyclic Geranylfarnesol and its Acetate by Squalene-hopene Cyclase from Alicyclobacillus acidocaldarius.

The enzymatic reactions of geranylfarnesol (8) and its acetate 9, classified as sesterterpenes (C25), using squalene-hopene cyclase (SHC) were investigated. The enzymatic reaction of 8 afforded 6/6-fused bicyclic 20, 6/6/6-fused tricyclic 21, and 6/6/6/6-fused tetracyclic compounds 22 and 23 as the main products (35% yield), whereas that of 9 afforded two 6/6/6-fused tricyclic compounds 24 and 25 in a high yield (76.3%) and a small amount (5.0%) of 26 (the acetate of 22). A significantly higher conversion of 9 indicates that the arrangement of the substrate in the reaction cavity changed. The lipophilic nature and/or the bulkiness of the acetyl group may have changed its binding with SHC, thus placing the terminal double bond of 9 in the vicinity of the DXDD motif of SHC, which is responsible for the proton attack on the double bond to initiate the polycyclization reaction. The results obtained for 8 are different to some extent than those reported by Shinozaki et al. The products obtained in this study were deprotonated compounds; however, the products reported by Shinozaki et al. were hydroxylated compounds.

Comparison of multi-enzyme and thermophilic bacteria on the hydrolysis of mariculture organic waste (MOW).

Mariculture organic waste (MOW) is rich in organic matter, which is a potential energy resource for anaerobic digestion. In order to enhance the anaerobic fermentation, the MOW was hydrolyzed by multi-enzyme and thermophilic bacteria. It was advantageous for soluble chemical oxygen demand (SCOD) release at MOW concentrations of 6 and 10 g/L with multi-enzyme and thermophilic bacteria pretreatments. For multi-enzyme, the hydrolysis was not obvious at substrate concentrations of 1 and 3 g/L, and the protein and carbohydrate increased with hydrolysis time at substrate concentrations of 6 and 10 g/L. For thermophilic bacteria, the carbohydrate was first released at 2-4 h and then consumed, and the protein increased with hydrolysis time. The optimal enzyme hydrolysis for MOW was determined by measuring the changes of SCOD, protein, carbohydrate, ammonia and total phosphorus, and comparing with acid and alkaline pretreatments.

Iron Acquisition in Mycobacterium avium subsp. paratuberculosis.

UNLABELLED: Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen. IMPORTANCE: Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro.

UNLABELLED: Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar_A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar_A2165, a putative transcriptional regulator. Mutants expressing the Mbar_A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar_A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri. IMPORTANCE: The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens.