Molecular and Functional Characterization of theRhodopseudomonas palustris No. 7 Phosphoenolpyruvate Carboxykinase Gene

ABSTRACT The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacteriumRhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of thepckA initiation codon and an upstream sequence similar to ς70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, thepckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.

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Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Journal of Bacteriology ◽

10.1128/jb.184.1.183-190.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 183-190 ◽

Cited By ~ 11

Author(s):

Michael J. Hynes ◽

Oliver W. Draht ◽

Meryl A. Davis

Keyword(s):

Carbon Source ◽

Aspergillus Nidulans ◽

Phosphoenolpyruvate Carboxykinase ◽

Carbon Sources ◽

Tca Cycle ◽

Northern Blotting ◽

Gene Encoding ◽

The Tca Cycle ◽

Key Enzyme ◽

Acetate Utilization

ABSTRACT Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.

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Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

Journal of Bacteriology ◽

10.1128/jb.181.3.849-857.1999 ◽

1999 ◽

Vol 181 (3) ◽

pp. 849-857 ◽

Cited By ~ 50

Author(s):

P. Aneja ◽

T. C. Charles

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Sinorhizobium Meliloti ◽

Transcriptional Start Site ◽

Consensus Sequence ◽

Start Codon ◽

Start Site ◽

Gene Encoding ◽

Reading Frame ◽

Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

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Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽

10.1093/genetics/147.4.1521 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1521-1531 ◽

Cited By ~ 2

Author(s):

Magne Østerås ◽

Shelley A P O'Brien ◽

Turlough M Finan

Keyword(s):

Sinorhizobium Meliloti ◽

Phosphoenolpyruvate Carboxykinase ◽

Root Nodules ◽

Genetic Regulation ◽

Carbon Sources ◽

Dicarboxylic Acids ◽

Phenotypic Analysis ◽

Gene Encoding ◽

Type Locus ◽

Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

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The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

Applied and Environmental Microbiology ◽

10.1128/aem.00054-18 ◽

2018 ◽

Vol 84 (13) ◽

Cited By ~ 3

Author(s):

Lei Zhang ◽

Muhang Li ◽

Qiqi Li ◽

Chaoqiong Chen ◽

Meng Qu ◽

...

Keyword(s):

Carbon Source ◽

Carbon Metabolism ◽

Carbon Sources ◽

Endophytic Bacterium ◽

Bacterial Attachment ◽

Gene Encoding ◽

Wheat Roots ◽

Host Colonization ◽

Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

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Chitin Utilization by the Insect-Transmitted Bacterium Xylella fastidiosa

Applied and Environmental Microbiology ◽

10.1128/aem.01036-10 ◽

2010 ◽

Vol 76 (18) ◽

pp. 6134-6140 ◽

Cited By ~ 37

Author(s):

Nabil Killiny ◽

Simone S. Prado ◽

Rodrigo P. P. Almeida

Keyword(s):

Carbon Source ◽

Biofilm Formation ◽

Xylella Fastidiosa ◽

Chitinolytic Activity ◽

Gene Encoding ◽

Reading Frame ◽

Biological Assays ◽

Chitinase A ◽

Transcriptional Changes

ABSTRACT Xylella fastidiosa is an insect-borne bacterium that colonizes xylem vessels of a large number of host plants, including several crops of economic importance. Chitin is a polysaccharide present in the cuticle of leafhopper vectors of X. fastidiosa and may serve as a carbon source for this bacterium. Biological assays showed that X. fastidiosa reached larger populations in the presence of chitin. Additionally, chitin induced phenotypic changes in this bacterium, notably increasing adhesiveness. Quantitative PCR assays indicated transcriptional changes in the presence of chitin, and an enzymatic assay demonstrated chitinolytic activity by X. fastidiosa. An ortholog of the chitinase A gene (chiA) was identified in the X. fastidiosa genome. The in silico analysis revealed that the open reading frame of chiA encodes a protein of 351 amino acids with an estimated molecular mass of 40 kDa. chiA is in a locus that consists of genes implicated in polysaccharide degradation. Moreover, this locus was also found in the genomes of closely related bacteria in the genus Xanthomonas, which are plant but not insect associated. X. fastidiosa degraded chitin when grown on a solid chitin-yeast extract-agar medium and grew in liquid medium with chitin as the sole carbon source; ChiA was also determined to be secreted. The gene encoding ChiA was cloned into Escherichia coli, and endochitinase activity was detected in the transformant, showing that the gene is functional and involved in chitin degradation. The results suggest that X. fastidiosa may use its vectors' foregut surface as a carbon source. In addition, chitin may trigger X. fastidiosa's gene regulation and biofilm formation within vectors. Further work is necessary to characterize the role of chitin and its utilization in X. fastidiosa.

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Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

10.1101/2020.11.13.381327 ◽

2020 ◽

Author(s):

Guiying Tang ◽

Pingli Xu ◽

Pengxiang Li ◽

Jieqiong Zhu ◽

Guangxia Chen ◽

...

Keyword(s):

Transcriptional Start Site ◽

Functional Characterization ◽

Regulatory Region ◽

Regulatory Elements ◽

Histochemical Staining ◽

Expression Vectors ◽

Upstream Sequence ◽

Functional Region ◽

Chromosomal Walking ◽

Storage Reserve

AbstractLEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5’ terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

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Production of Poly(3-Hydroxybutyrate) by Haloarcula, Halorubrum, and Natrinema Haloarchaeal Genera Using Starch as a Carbon Source

Archaea ◽

10.1155/2021/8888712 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Fatma Karray ◽

Manel Ben Abdallah ◽

Nidhal Baccar ◽

Hatem Zaghden ◽

Sami Sayadi

Keyword(s):

Carbon Source ◽

Carbon Sources ◽

Pcr Amplification ◽

Halophilic Archaea ◽

Hypersaline Lake ◽

Rrna Gene ◽

Plastic Pollution ◽

Promising Alternative ◽

Cell Factories ◽

Phb Production

Microbial production of bioplastics, derived from poly(3-hydroxybutyrate) (PHB), have provided a promising alternative towards plastic pollution. Compared to other extremophiles, halophilic archaea are considered as cell factories for PHB production by using renewable, inexpensive carbon sources, thus decreasing the fermentation cost. This study is aimed at screening 33 halophilic archaea isolated from three enrichment cultures from Tunisian hypersaline lake, Chott El Jerid, using starch as the sole carbon source by Nile Red/Sudan Black staining and further confirmed by PCR amplification of phaC and phaE polymerase genes. 14 isolates have been recognized as positive candidates for PHA production and detected during both seasons. The identification of these strains through 16S rRNA gene analyses showed their affiliation to Halorubrum, Natrinema, and Haloarcula genera. Among them, three PHB-producing strains, CEJ34-14, CEJ5-14, and CEJ48-10, related to Halorubrum chaoviator, Natrinema pallidum, and Haloarcula tradensis were found to be the best ones reaching values of 9.25, 7.11, and 1.42% of cell dry weight (CDW), respectively. Our findings highlighted that Halorubrum, Natrinema, and Haloarcula genera were promising candidates for PHB production using soluble starch as a carbon source under high salinity (250 g L-1 NaCl).

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Cloning and functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

PLoS ONE ◽

10.1371/journal.pone.0242949 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0242949

Author(s):

Guiying Tang ◽

Pingli Xu ◽

Pengxiang Li ◽

Jieqiong Zhu ◽

Guangxia Chen ◽

...

Keyword(s):

Transcriptional Start Site ◽

Functional Characterization ◽

Regulatory Region ◽

Regulatory Elements ◽

Histochemical Staining ◽

Expression Vectors ◽

Upstream Sequence ◽

Functional Region ◽

Chromosomal Walking ◽

Storage Reserve

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5’ terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

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Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2453-2460.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2453-2460 ◽

Cited By ~ 73

Author(s):

Ronald P. de Vries ◽

Hetty C. van den Broeck ◽

Ester Dekkers ◽

Paloma Manzanares ◽

Leo H. de Graaff ◽

...

Keyword(s):

Amino Acids ◽

Aspergillus Niger ◽

Signal Sequence ◽

Expression Patterns ◽

Carbon Sources ◽

Gene Encoding ◽

Calculated Molecular Mass ◽

Reading Frame ◽

Polymeric Compounds ◽

Elevated Expression

ABSTRACT A gene encoding a third α-galactosidase (AglB) fromAspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includesA. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA,aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.

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A Disjointed Pathway for Malonate Degradation by Rhodopseudomonas palustris

Applied and Environmental Microbiology ◽

10.1128/aem.00631-20 ◽

2020 ◽

Vol 86 (11) ◽

Author(s):

Zhaobao Wang ◽

Qifeng Wen ◽

Caroline S. Harwood ◽

Bo Liang ◽

Jianming Yang

Keyword(s):

Carbon Source ◽

Dicarboxylic Acid ◽

Slow Growth ◽

Rhodopseudomonas Palustris ◽

Carbon Sources ◽

Value Added ◽

Poor Growth ◽

Content Type ◽

Phototrophic Bacterium ◽

Malonyl Coa

ABSTRACT The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl-CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure. IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently.

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