Isolation, oxygen sensitivity, and virulence of NADH oxidase mutants of the anaerobic spirochete Brachyspira (Serpulina) hyodysenteriae, etiologic agent of swine dysentery.

Brachyspira (Serpulina) hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no. U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmDeltaNOX and pKmDeltaNOX, were used to transform wild-type B. hyodysenteriae B204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-type nox genes in these strains had been replaced, through allelic exchange, by the inactivated nox gene containing cat or kan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that both nox mutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of both nox mutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, both nox mutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protect B. hyodysenteriae cells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.     

NADH oxidase ofThermus thermophilus HB8 overproduced fromEscherichia coli

Summary An NADH oxidase purified from the extreme thermophileThermus thermophilus HB8 is a monomeric flavoprotein with a 1 1 ratio of flavin-adenine dinucleotide (FAD) to the polypeptide chain. It catalyzes in vitro the oxidation of reduced NADH or NADPH with the formation of H₂O₂. The gene encoding the NADH oxidase fromT. thermophilus HB8 was cloned, and its nucleotide sequence was determined. The molecular mass of 22,749 Da, as deduced from thenox gene, agrees with that of the purified NADH oxidase fromT. thermophilus HB8, as estimated by mass spectrometry. Thenox gene does not contain a GX₄GK consensus sequence typical for nucleotide binding proteins. Thenox gene was overexpressed inEscherichia coli, and a protocol for the rapid purification of theE. coli-borneT. thermophilus NADH oxidase or its His₆-tagged analogue was developed by using thermal denaturation step and affinity chromatography.     

Brachyspira (Serpulina) hyodysenteriae gyrB Mutants and Interstrain Transfer of Coumermycin A1 Resistance

To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, the gyrB gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A₁-resistant (Cn^r) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A₁/ml. The coumermycin A₁ MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cn^r strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly₇₈ to Ser (two strains), Gly₇₈ to Cys, and Thr₁₆₆ to Ala. When Cn^r strain 435A (Gly₇₈ to Ser) and Cm^r Km^r strain SH (ΔflaA1::cat Δnox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A₁, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven Cn^r Km^r Cm^r strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cn^r Km^r Cm^r cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with a gyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.     

Identification of a Linked Set of Genes in Serpulina hyodysenteriae (B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein).     

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.     

Hydrogen production by Escherichia coli containing a cloned hydrogenase gene from Citrobacter freundii

Two hydrogenase genes of Citrobacter freundii complementing different Escherichia coli hyd mutations were cloned on the multicopy-plasmid pBR322. Recombinant plasmids pCBH2 and pCFH1 were obtained. Since hydrogenase activities of E. coli transformant HK-8 (pCBH2) and HK-7 (pCFH1) were much the same as E. coli C600 (wild type cells), the reduction in DNA size of recombinant plasmid pCBH2 (10.7 kb) was investigated. Reduced recombinant plasmids pCBH4 (6.2 kb) and pCBH6 (5.7 kb) were obtained, and a hydrogenase gene was found to be located on the 2.35 kb fragment between AvaI and EcoRI sites. Hydrogenase activity and hydrogen-evolving activity of E. coli HK-8 (pCBH4 or pCBH6) from sodium formate, sodium pyruvate or glucose were approximately 2-fold higher than those of E. coli C600 (wild type cells).On the other hand, a reduced recombinant plasmid pCBH10 (6.0 kb), which contained the adjacent DNA fragment (2.15 kb) to a hydrogenase gene, was obtained. Hydrogenase activity of E. coli C600 harboring pCBH10 was half that of E. coli C600. From these results we estimate that in plasmid pCBH2, the repressor gene suppressing the synthesis of hydrogenase might have been cloned together with a hydrogenase gene.     

High-Level Acetaldehyde Production in Lactococcus lactis by Metabolic Engineering

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.     

细菌nox基因研究进展

细菌nox基因编码合成一种含核黄素的NADH氧化酶,NADH氧化酶可催化双原子氧还原为H2O2或H2O,同时将NADH氧化为NAD+。该反应发生在多种代谢途径中,从而对细菌的氧化应激、菌膜形成、毒力调控及代谢产物生成等生理生化过程产生一系列影响。目前对高等动植物体中的nox基因及其编码的NADH氧化酶已有较深入的研究,但近年来一些研究表明,细菌nox基因的功能及作用通路与动植物体存在较大差异,因此,有必要详细了解细菌中nox基因和NADH氧化酶的具体作用机制及其对细胞产生的影响。综合分析近年来细菌nox基因及NADH氧化酶的研究成果,结合我们的研究,对目前存在的问题和未来的发展进行综述。     

The Complete Genome Sequence of the Pathogenic Intestinal Spirochete Brachyspira pilosicoli and Comparison with Other Brachyspira Genomes

Background

The anaerobic spirochete Brachyspira pilosicoli colonizes the large intestine of various species of birds and mammals, including humans. It causes “intestinal spirochetosis”, a condition characterized by mild colitis, diarrhea and reduced growth. This study aimed to sequence and analyse the bacterial genome to investigate the genetic basis of its specialized ecology and virulence.

Methodology/Principal Findings

The genome of B. pilosicoli 95/1000 was sequenced, assembled and compared with that of the pathogenic Brachyspira hyodysenteriae and a near-complete sequence of Brachyspira murdochii. The B. pilosicoli genome was circular, composed of 2,586,443 bp with a 27.9 mol% G+C content, and encoded 2,338 genes. The three Brachyspira species shared 1,087 genes and showed evidence of extensive genome rearrangements. Despite minor differences in predicted protein functional groups, the species had many similar features including core metabolic pathways. Genes distinguishing B. pilosicoli from B. hyodysenteriae included those for a previously undescribed bacteriophage that may be useful for genetic manipulation, for a glycine reductase complex allowing use of glycine whilst protecting from oxidative stress, and for aconitase and related enzymes in the incomplete TCA cycle, allowing glutamate synthesis and function of the cycle during oxidative stress. B. pilosicoli had substantially fewer methyl-accepting chemotaxis genes than B. hyodysenteriae and hence these species are likely to have different chemotactic responses that may help to explain their different host range and colonization sites. B. pilosicoli lacked the gene for a new putative hemolysin identified in B. hyodysenteriae WA1. Both B. pilosicoli and B. murdochii lacked the rfbBADC gene cluster found on the B. hyodysenteriae plasmid, and hence were predicted to have different lipooligosaccharide structures. Overall, B. pilosicoli 95/1000 had a variety of genes potentially contributing to virulence.

Conclusions/Significance

The availability of the complete genome sequence of B. pilosicoli 95/1000 will facilitate functional genomics studies aimed at elucidating host-pathogen interactions and virulence.     

Cloning and characterization of a lysine decarboxylase gene from Hafnia alvei

Summary A lysine decarboxylase (LDC) gene from Hafnia alvei was cloned in the Escherichia coli strain HB101. A gene bank consisting of 2,000 clones, carrying recombinant plasmids with large DNA fragments of H. alvei integrated in the BamH1 site of pBR322, was screened for LDC activity by a colony filter radioimmunoassay. The gene bank yielded clone 462 expressing high LDC activity with the presence of a plasmid carrying a 7.5 kb insert of H. alvei. Two LDC-positive subclones derived from 462 with inserts of 2.9 and 3.3 kb were sequenced by the shotgun method. An open reading frame for a 83 K protein with 739 amino acids was determined as the coding region for the LDC. The identification of this reading frame as the true reading frame of the H. alvei LDC gene and its similarities with LDC of E. coli are described. The use of the cloned gene for the transformation of plant cells is discussed.     

Genetic Diversity of Bile Salt Hydrolases Among Human Intestinal Bifidobacteria

Attachment of Brachyspira hyodysenteriae to intestinal epithelial cell lines and its possible mediation by outer membrane proteins (OMPs) of the spirochete were examined. Different B. hyodysenteriae serotypes were shown to adhere to rat and swine intestinal epithelial cells (IEC-18 and IPEC-J2) in vitro but not to the human rectal tumor cell line (HRT-18). Adherence of strain B204 to IPEC-J2 cells was reduced by rOMP-specific antisera in amounts of 29 % (anti-rBhlp29.7), 59 % (anti-rBhlp16), 70 % (anti-rBhmp39h), and 74 % (anti-rBhmp39h), respectively. By use of pooled antisera against Bhlp16 and Bhmp39f inhibition rates of the other serotypes ranged from 53 to 91 %. In a western blot assay OMPs of all serotypes but one were detected by the respective rOMP antisera. Altogether the results indicated that OMPs of B. hyodysenteriae displayed a serotype overlapping antigenicity and mediated adherence of the spirochetes to animal cell cultures.     

Cloning and characterization of the gene, speC, for pyrogenic exotoxin type C from Streptococcus pyogenes

Summary The structural gene of streptococcal pyrogenic exotoxin type C (SPE C) was cloned from the chromosome of Streptococcus pyogenes strain T18P into Escherichia coli using pBR328 as the vector plasmid. Subcloning enabled the localization of the gene (speC) to a 1.7 kb fragment. Partially purified E. coli-derived SPE C and purified streptococcal-derived SPE C, were shown to have the same molecular weight (23 800) and biological activities. A DNA probe, prepared from cloned speC, cross-hybridized with the structural genes of SPE A and SPE B indicating relatedness at the nucleotide level. The speC-derived probe also hybridized to a fragment of CS112 bacteriophage DNA containing the phage attachment site.     

Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product n required for assembly of all c-type cytochromes, including cytochrome c1

A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome C₅₅₀, but the membrane-bound holocytochromes c₁ and C₅₅₂ were present at 5-10% of the levels observed in wild-type ceils, DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb Bam HI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DPI08 and the B. japonicum cycH mutant C0X3, especially with respect to cytochrome c₁ synthesis, suggest that the cycH gene product may be an assembly factor.     

Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum

A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg²⁺ in the γ-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.     

Molecular studies on thiaminase I

The thiaminase I gene of Bacillus thiaminolyticus was cloned on a 1.6 kb DNA fragment (enzyme molecular weight 42 000), and was expressed in both Escherichia coli and Bacillus subtilis. When a selection drug was absent, the plasmid was maintained stably for approx. 100 generations in wild-type E. coli. Instability of the thiaminase gene was demonstrated in the thiamin pyrophosphate-requiring mutant of E. coli from which the plasmid was deleted rapidly. Wild-type E. coli accumulated the enzyme in its periplasm. A method for the detection of thiaminase I enzyme in SDS-polyacrylamide gel was developed. Thiaminase I of B. thiaminolyticus was found to exist in two sizes, 44 and 42 kDa, among different strains. Moreover, thiaminase of 42 kDa became approximately 41 kDa after a long-term culture, most likely because of the action of proteinases. Thiaminase expressed in E. coli from a thiaminase-positive recombinant plasmid was 42 kDa, and showed the same mobility on SDS-polyacrylamide gele electrophoresis as the enzyme isolated from the young culture of the parent strain of B. thiaminolyticus used for cloning. This value was, therefore, considered to represent intact thiaminase that had escaped from the attack of bacilli proteinases.     

Conversion of sub-megasized DNA to desired structures using a novel Bacillus subtilis genome vector

A novel genome vector using the 4215 kb Bacillus subtilis genome provides for precise target cloning and processing of the cloned DNA to the desired structure. Each process highly dependent on homologous recombination in the host B.subtilis is distinguished from the other cloning systems. A 120 kb mouse jumonji (jmj) genomic gene was processed in the genome vector to give a series of truncated sub-megasized DNA. One of these truncated segments containing the first intron was copied in a plasmid by a recombinational transfer method developed for B.subtilis. DNA manipulation previously considered difficult is argued with respect to DNA size and accuracy.