Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange.

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.     

Cloning, sequencing and expression of the acylneuraminate lyase gene from Clostridium perfringens A99

The acylneuraminate lyase gene from Clostridium perfringens A99 was cloned on a 3.3 kb HindIII DNA fragment identified by screening the chromosomal DNA of this species by hybridization with an oligonucleotide probe that had been deduced from the N-terminal amino acid sequence of the purified protein, and another probe directed against a region that is conserved in the acylneuraminate lyase gene of Escherichia coli and in the putative gene of Clostridium tertium. After cloning, three of the recombinant clones expressed lyase activity above the background of the endogenous enzyme of the E. coli host. The sequenced part of the cloned fragment contains the complete acylneuraminate lyase gene (ORF2) of 864 bp that encodes 288 amino acids with a calculated molecular weight of 32.3 kDa. The lyase structural gene follows a non-coding region with an inverted repeat and a ribosome binding site. Upstream from this regulatory region another open reading frame (ORF1) was detected. The 3′-terminus of the lyase structural gene is followed by a further ORF (ORF3). A high homology was found between the amino acid sequences of the sialate lyases from Clostridium perfringens and Haemophilus influenzae (75% identical amino acids) or Trichomonas vaginalis (69% identical amino acids), respectively, whereas the similarity to the gene from E. coli is low (38% identical amino acids). Based on our new sequence data, the ‘large’ sialidase gene and the lyase gene of C. perfringens are not arranged next to each other on the chromosome of this species. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of a novel ferredoxin with N-terminal extension from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824

A gene (CAC2657) encoding a ferredoxin (EFR1) from the strictly anaerobic soil bacterium Clostridium acetobutylicum was cloned and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 27 kDa that incorporates 2[4Fe–4S] clusters. An extended N-terminal region of 187 amino acid (aa) residues precedes ferredoxin domain. The EFR1 expressed in E. coli is a trimeric protein. The iron and sulfur content of the reconstituted protein agrees with that expected of a trimeric form of the protein. The ferredoxin domain of EFR1 is closely related to ferredoxin of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 0.62 As for the Cα atoms of the generated 3D simulation model. In cultures of C. acetobutylicum the efr1 gene shows higher relative expression on induction with Trinitrotoluene (TNT) compared to that from uninduced control cultures.     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

C4-dicarboxylate carriers and sensors in bacteria.

Bacteria contain secondary carriers for the uptake, exchange or efflux of C4-dicarboxylates. In aerobic bacteria, dicarboxylate transport (Dct)A carriers catalyze uptake of C4-dicarboxylates in a H(+)- or Na(+)-C4-dicarboxylate symport. Carriers of the dicarboxylate uptake (Dcu)AB family are used for electroneutral fumarate:succinate antiport which is required in anaerobic fumarate respiration. The DcuC carriers apparently function in succinate efflux during fermentation. The tripartite ATP-independent periplasmic (TRAP) transporter carriers are secondary uptake carriers requiring a periplasmic solute binding protein. For heterologous exchange of C4-dicarboxylates with other carboxylic acids (such as citrate:succinate by CitT) further types of carriers are used. The different families of C4-dicarboxylate carriers, the biochemistry of the transport reactions, and their metabolic functions are described. Many bacteria contain membraneous C4-dicarboxylate sensors which control the synthesis of enzymes for C4-dicarboxylate metabolism. The C4-dicarboxylate sensors DcuS, DctB, and DctS are histidine protein kinases and belong to different families of two-component systems. They contain periplasmic domains presumably involved in C4-dicarboxylate sensing. In DcuS the periplasmic domain seems to be essential for direct interaction with the C4-dicarboxylates. In signal perception by DctB, interaction of the C4-dicarboxylates with DctB and the DctA carrier plays an important role.     

Cloning and characterization of a maltotriose-producing α-amylase gene from <Emphasis Type="Italic">Thermobifida fusca</Emphasis>

The gene (tfa), encoding a maltotriose-producing α-amylase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1,815 base pairs and encodes a protein of 605 amino acids. The base composition of the tfa coding sequence is 69% G+C and the protein has a predicted pI value of 5.5. The deduced amino acid sequence of the tfa amylase exhibited a high degree of similarity with amylases from Thermomonospora curvata and Streptomyces amylases. The purified amylase could be detected as a single band of about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The properties of purified amylase from the E. coli transformant are similar to that of an amylase purified from the original T. fusca NTU22.     

Cloning and expression of the Neisseria meningitidis 5C outer membrane protein

The 5C outer membrane protein, one of the N. meningitidis class 5 proteins, was preferably expressed in bacteria isolated from the nasopharynx and its role in adhering to the mucosal cells and invading them as well as the development of anti-5C antibodies in healthy carriers was demonstrated. Anti-5C monoclonal antibodies are bactericidal in the presence of the human complement. The immunodominant region of the 5C protein is highly conserved among the different strains of N. meningitidis, and the opc gene, which encodes the protein, does not seem to show antigenic variations. Here the isolation of the opc gene from the Cuban strain B:4:P1.15 by PCR (Polymerase Chain Reaction) is presented. Under the regulation of the tryptophan promoter, the gene was cloned and sequenced in E. coli with a high level of expression and fused to the amino-terminal end of the interleukin-2 gene. In the dot-blot experiments, the presence of the gene in those strains which did not express the protein in the whole cell ELISA was also detectable.     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

C4-dicarboxylates and l-aspartate utilization by Escherichia coli K-12 in the mouse intestine: l-aspartate as a major substrate for fumarate respiration and as a nitrogen source

C4-dicarboxylates, such as fumarate, l -malate and l -aspartate represent substrates for anaerobic growth of Escherichia coli by fumarate respiration. Here, we determined whether C4-dicarboxylate metabolism, as well as fumarate respiration, contribute to colonization of the mammalian intestinal tract. Metabolite profiling revealed that the murine small intestine contained high and low levels of l -aspartate and l -malate respectively, whereas fumarate was nearly absent. Under laboratory conditions, addition of C4-dicarboxylate at concentrations corresponding to the levels of the C4-dicarboxylates in the small intestine (2.6 mmol kg⁻¹ dry weight) induced the dcuBp-lacZ reporter gene (67% of maximal) in a DcuS-DcuR-dependent manner. In addition to its role as a precursor for fumarate respiration, l -aspartate was able to supply all the nitrogen required for anaerobically growing E. coli. DcuS-DcuR-dependent genes were transcribed in the murine intestine, and mutants with defective anaerobic C4-dicarboxylate metabolism (dcuSR, frdA, dcuB, dcuA and aspA genes) were impaired for colonizing the murine gut. We conclude that l -aspartate plays an important role in providing fumarate for fumarate respiration and supplying nitrogen for E. coli in the mouse intestine.     

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen-metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese-containing SOD ([Mn]-SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]-SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in-frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]-SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]-SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur- and Fnr-binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis- responsive activators in H. influenzae gene expression.     

The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid

Shufflon DNA rearrangement selects one of seven PilV proteins with different C-terminal segments, which then becomes a minor component of the thin pili of Escherichia coli strains bearing the plasmid R64. The PilV proteins determine the recipient specificity in liquid matings. A recipient Escherichia coli K-12 strain was specifically recognized by the PilVA′, -C, and -C′ proteins, while E. coli B was recognized only by the PilVA′ protein. To identify specific PilV receptors in the recipient bacterial cells, R64 liquid matings were performed using various E. coli K-12 waa (rfa) mutants and E. coli B transformants as recipient cells. E. coli K-12 waa mutants lack receptors for specific PilV proteins. E. coli B cells carrying waaJ or waaJKL genes of E. coli K-12 were recognized by donors expressing the PilVC′ protein or the PilVC and -C′ proteins, respectively, in addition to the PilVA′ protein. Addition of E. coli K-12 or B lipopolysaccharide (LPS) specifically inhibited liquid matings. We conclude that the PilV proteins of the thin pili of R64-bearing donors recognize LPS molecules located on the surface of various recipient bacterial cells in liquid matings. Received: 2 September 1999 / Accepted: 18 November 1999     

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Induction of haemolytic activity in Escherichia coli by the slyA gene product

The Salmonella typhimurium protein SlyA_ST, originally described as a cytolysin, shows sequence similarities to several known bacterial regulatory proteins. A homologue to the slyA_St gene has been localised to min 37 of the Eschericia coli K-12 chromosome and has been designated slyA_EC When introduced in trans on a plasmid, the slyA_EC gene conferred a haemolytic phenotype on wild-type but not clyA-knockout strains of E. coli K-12. The clyA gene encodes a novel haemolysin that is not expressed by wild-type E. coli under tested laboratory conditions. Western and Northern blot analyses, and DNA-band-shift assays support a model whereby the SlyA_EC protein activates clyA expression by binding to the clyA promoter region, thereby supporting the sequence similarity data in suggesting that SlyA_ST is a haemolysin activator rather than being a haemolysin per se.     

The ORF1 of the gentamicin-resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5'-phosphosulphate kinase

The gentamicin-resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine-5′-phosphosulphate (APS) kinase. APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3′-hydroxyl group of APS. The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3′-hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E. coli APS kinase. In extracts of pseudomonad cells we have also detected a higher molecular mass (70 kDa) protein that cross-reacts with an anti-E. coli APS kinase antibody. This cross-reactive protein is also present in Pseudomonas strains lacking the gentamicin-resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ-encoded high-molecular-weight APS kinase present in Rhizobium meliloti. Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E. coli, as is E. coli APS kinase. However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1 -encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.     

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker

We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase – a selectable nuclear marker – in Chlamydomonas reinhardtii. The 5′ end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8φ3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed. Received: 2 June 1998 / Accepted: 25 September 1998     

Anaerobic growth of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.     

Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.