Cloning, Sequence, and Expression of the L-(+) Lactate Dehydrogenase of Streptococcus bovis

The ldh gene encoding the fructose-1,6-diphosphate-dependent L-(+) lactate dehydrogenase from the ruminal bacterium Streptococcus bovis was cloned and sequenced. A genomic library of S. bovis JB1 DNA was constructed in lambda ZAP II and screened by use of a heterologous probe derived from the cloned Streptococcus mutans ldh gene. Several clones were isolated that contained a common 2.9-kb fragment as determined by restriction analysis. Nucleotide sequence analysis revealed a 987-bp open reading frame with extensive homology to Streptococcus thermophilus and S. mutans ldh nucleic acid and amino acid sequences. Expression of the cloned S. bovis ldh gene in Escherichia coli was confirmed by the ability to complement the ldh mutation of E. coli FMJ39, by using an in-gel activity screen and by enzymatic assay. Increased LDH activity was observed in S. bovis JB1 containing the cloned ldh genes on a multicopy plasmid. Received: 15 October 1996 / Accepted: 3 December 1996     

Lactate dehydrogenase gene variability among predominant lactate utilizing ruminal bacteria

The inter- and intraspecies variability of lactate dehydrogenase (ldh) gene was determined among the predominant ruminal lactate utilizing bacteria. Nearly complete nucleotide sequences of ldh gene, encoding NAD-dependent lactate dehydrogenase of three Megasphaera elsdenii and six Selenomonas ruminantium strains, were obtained and compared. Phylogenetic analyses revealed a limited variability between the ldh sequences studied. The majority of differences observed were silent mutations at the 3rd position of codons. Surprisingly, the intraspecies diversity of the ldh gene among S. ruminantium isolates was higher than the interspecies level between S. ruminantium and M. elsdenii, which strongly suggests the possibility of acquisition of this gene by horizontal gene transfer.     

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization

Summary From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr⁺ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K. pneumoniae.     

Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3

A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 by EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD⁺-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or scrotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.     

Cloning and Characterization of a 4-Hydroxyphenylacetate 3-Hydroxylase From the Thermophile <Emphasis Type="Italic">Geobacillus</Emphasis> sp. PA-9

A 4-hydroxyphenylacetic acid (4-HPA) hydroxylase-encoding gene, on a 2.7-kb genomic DNA fragment, was cloned from the thermophile Geobacillus sp. PA-9. The Geobacillus sp. PA-9 4-HPA hydroxylase gene, designated hpaH, encodes a protein of 494 amino acids with a predicted molecular mass of 56.269 Da. The deduced amino-acid sequence of the hpaH gene product displayed <30% amino-acid sequence identity with the larger monooxygenase components of the previously characterized two-component 4-HPA 3-hydroxylases from Escherichia coli W and Klebsiella pneumoniae M5a1. A second oxidoreductase component was not present on the 2.7-kb genomic DNA fragment. The deduced amino-acid sequence of a second C-terminal truncated open reading frame, designated hpaI, exhibited homology to extradiol oxygenases and displayed the highest amino-acid sequence identity (43%) with the 3,4-dihydroxyphenylacetate 2,3-dioxygenase of Arthrobacter globiformis, encoded by mndD. These results, along with catalytic activity observed in crude intracellular extracts prepared from Escherichia coli cells expressing hpaH, is in support of a role for hpaH in the 4-HPA degradative pathway of Geobacillus sp. PA-9.     

Molecular analysis of thescrA andscrB genes fromKlebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme IIScr of the phosphotransferase system and a sucrose-6-phosphate invertase

TheKlebsiella pneumoniae genesscrA andscrB are indispensable for sucrose (Scr) utilisation. GenescrA codes for an Enzyme II^Scr (II^Scr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), whilescrB encodes a sucrose 6-phosphate specific invertase. A 3.7 kbscrAB DNA fragment has been cloned fromK. pneumoniae and expressed inEscherichia coli. Its nucleotide sequence was determined and the coding regions forscrA (1371 bp) andscrB (1401 bp) were identified by genetic complementation, enzyme activity tests and radiolabelling of the gene products. In addition, the nucleotide sequence of thescrB gene from the conjugative plasmid pUR400 isolated fromSalmonella typhimurium was also determined and errors in the previously published sequence of thescrA gene of pUR400 were corrected. Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes. Based on the analysis of seven II^Scr proteins, a hypothetical model of the secondary structure of II^Scr is proposed.     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric -ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Sequencing and analysis of aMycoplasma gallisepticum A5969 chromosome region containing the S10 andrrn23-5 operons

A 20,115-nt region of theMycoplasma gallisepticum A5959 genome was sequenced (GenBank accession no. AF036708). The region contains therrn23-5 and S10 operons, the lactate dehydrogenase gene, and two open reading frames (ORF293 and ORF129/ORF171) coding for proteins of unknown function. Therrn23-5 operon includes genes for 23S and 5S rRNAs. The S10 operon includes genes for 20 ribosomal proteins, Sec Y transport protein, adenylate kinase, and methionine aminopeptidase, and lacks theinfA-rpl36-rps13-rpoA-rpl17 genes found in the S10 operon ofM. genitalium, M. pneumoniae, andBacillus subtilis. The product ofM. gallisepticum ldh is equally similar to the corresponding proteins of mycoplasmata andB. subtilis but contains only a part of the motif characteristic of the active center of lactate dehydrogenases. The chromosome region adjacent to the sequenced one containsuvrA,nrdE,nrdF, andptsI.     

Differentiation of Chlamydia Species by Combined Use of Polymerase Chain Reaction and Restriction Endonuclease Analysis

To differentiate Chlamydia spp., a primer pair designed to generate a genus-specific region of the major outer membrane protein (MOMP) gene was used in a PCR to amplify a single DNA fragment of 245-259 bp. In the PCR, the expected single DNA fragment was amplified from strains of Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, respectively. By restriction endonuclease analysis with AluI and PvuII, the amplified products exhibited four distinct patterns, corresponding to the four species. It is, therefore, concluded that one-step PCR followed by restriction endonuclease analysis as described in this study could be a valuable method for the detection and differentiation of Chlamydia species.     

The nifHDK genes are contiguous with a nifA-like regulatory gene in Rhodobacter capsulatus

Summary A 15.2 kb DNA fragment was isolated from Rhodobacter capsulatus (ex. Rhodopseudomonas capsulata), which was able to complement mutations both in a nifA-like regulatory gene and in the nifH gene. Physical mapping of this fragment revealed that the nifA-like gene was adjacent to, and downstream from, the nifHDK operon. Hybridization experiments were carried out using a cloned Klebsiella pneumoniae DNA fragment containing nifA and the flanking portions of nifB and nifL. This fragment failed to hybridize with a 2.15 kb HindIII fragment of R. capsulatus DNA containing the nifA-like gene, but hybridized instead with a 2.6 kb EcoRI fragment adjacent to the nifA-like gene. The homologous region was found to be located within the K. pneumoniae nifB gene. The adjacent 2.6 kb and 2.15 kb fragments also hybridized with each other, indicating the presence of repeated sequences in this region.     

Role of the pyruvate metabolic network on carbohydrate metabolism and virulence in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that must adapt to unique nutritional environments in several host niches. The pneumococcus can metabolize a range of carbohydrates that feed into glycolysis ending in pyruvate, which is catabolized by several enzymes. We investigated how the pneumococcus utilizes these enzymes to metabolize different carbohydrates and how this impacts survival in the host. Loss of ldh decreased bacterial burden in the nasopharynx and enhanced bacteremia in mice. Loss of spxB, pdhC or pfl2 decreased bacteremia and increased host survival. In glucose or galactose, loss of ldh increased capsule production, whereas loss of spxB and pdhC reduced capsule production. The pfl2 mutant exhibited reduced capsule production only in galactose. In glucose, pyruvate was metabolized primarily by LDH to generate lactate and NAD⁺ and by SpxB and PDHc to generate acetyl-CoA. In galactose, pyruvate metabolism was shunted toward acetyl-CoA production. The majority of acetyl-CoA generated by PFL was used to regenerate NAD⁺ with a subset used in capsule production, while the acetyl-CoA generated by SpxB and PDHc was utilized primarily for capsule biosynthesis. These data suggest that the pneumococcus can alter flux of pyruvate metabolism dependent on the carbohydrate present to succeed in distinct host niches.     

Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme

The spi gene of Streptococcus pneumoniae was cloned and its nucleotide sequence was determined. It encodes a protein of 204 amino acids that is homologous to bacterial signal peptidase I proteins. The S. pneumoniae protein contains all of the conserved amino acid sequence motifs previously identified in this enzyme from both prokaryotic and eukaryotic sources. Sequence comparisons revealed several additional motifs characteristic of the enzyme. The cloned S. pneumoniae gene complemented an Escherichia coli mutant defective in its leader peptidase gene. Expression of the spi gene in S. pneumoniae appeared to be essential for viability. The cloned gene was shown to produce a polypeptide of approximately 20 kDa. Overproduction of the S. pneumoniae spi gene in an E. coli expression system gave a native protein product, soluble in the presence of a non-ionic detergent, which should be amenable to structural determination.     

Mycoplasma pneumoniae DNA gyrase genes

We have identified a clone from a λEMBL3 library containing a 19kb insert of Mycoplasma pneumoniae DNA which includes the genes that encode both subunits of DNA gyrase. The gyrB gene and the 5’end of the gyrA gene have been subcloned into M13. The gryB gene is 1953bp in length and overlaps the gryA gene by a single base. The nucleotide sequence of these subclones has significant homology to previously reported gyrase genes. In terms of the size of gyrB gene and its proximity to the gyrA gene, M. pneumoniae is more similar to Bacillus subtilis than to Escherichia coli.     

Native xylose-inducible promoter expands the genetic tools for the biomass-degrading,extremely thermophilic bacterium Caldicellulosiruptor bescii

Regulated control of both homologous and heterologous gene expression is essential for precise genetic manipulation and metabolic engineering of target microorganisms. However, there are often no options available for inducible promoters when working with non-model microorganisms. These include extremely thermophilic, cellulolytic bacteria that are of interest for renewable lignocellulosic conversion to biofuels and chemicals. In fact, improvements to the genetic systems in these organisms often cease once transformation is achieved. This present study expands the tools available for genetically engineering Caldicellulosiruptor bescii, the most thermophilic cellulose-degrader known growing up to 90 °C on unpretreated plant biomass. A native xylose-inducible (P _xi ) promoter was utilized to control the expression of the reporter gene (ldh) encoding lactate dehydrogenase. The P _xi -ldh construct resulted in a both increased ldh expression (20-fold higher) and lactate dehydrogenase activity (32-fold higher) in the presence of xylose compared to when glucose was used as a substrate. Finally, lactate production during growth of the recombinant C. bescii strain was proportional to the initial xylose concentration, showing that tunable expression of genes is now possible using this xylose-inducible system. This study represents a major step in the use of C. bescii as a potential platform microorganism for biotechnological applications using renewable biomass.

     

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>, <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.