Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW

Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H?) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose·L?1, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO?), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H? production was 14.2 mmol·(L culture)?1 and the total production of ethanol was 0.4 mmol·(L culture)?1. The maximum yield of H? was 1.3 mol·(mol glucose equivalent)?1 at a carbon recovery of 94%. The specific production rates of H?, CO?, and ethanol were 0.45, 0.13, and 0.003 mol·h?1·(g dry cell mass)-1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate-synthesizing clostridia.     

Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T.?thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (>?0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.     

Insights into electron flux through manipulation of fermentation conditions and assessment of protein expression profiles in Clostridium thermocellum

While annotation of the genome sequence of Clostridium thermocellum has allowed predictions of pathways catabolizing cellobiose to end products, ambiguities have persisted with respect to the role of various proteins involved in electron transfer reactions. A combination of growth studies modulating carbon and electron flow and multiple reaction monitoring (MRM) mass spectrometry measurements of proteins involved in central metabolism and electron transfer was used to determine the key enzymes involved in channeling electrons toward fermentation end products. Specifically, peptides belonging to subunits of ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase (NFOR) were low or below MRM detection limits when compared to most central metabolic proteins measured. The significant increase in H₂ versus ethanol synthesis in response to either co-metabolism of pyruvate and cellobiose or hypophosphite mediated pyruvate:formate lyase inhibition, in conjunction with low levels of ferredoxin-dependent hydrogenase and NFOR, suggest that highly expressed putative bifurcating hydrogenases play a substantial role in reoxidizing both reduced ferredoxin and NADH simultaneously. However, product balances also suggest that some of the additional reduced ferredoxin generated through increased flux through pyruvate:ferredoxin oxidoreductase must be ultimately converted into NAD(P)H either directly via NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase (NfnAB) or indirectly via NADPH-dependent hydrogenase. While inhibition of hydrogenases with carbon monoxide decreased H₂ production 6-fold and redirected flux from pyruvate:ferredoxin oxidoreductase to pyruvate:formate lyase, the decrease in CO₂ was only 20 % of that of the decrease in H₂, further suggesting that an alternative redox system coupling ferredoxin and NAD(P)H is active in C. thermocellum in lieu of poorly expressed ferredoxin-dependent hydrogenase and NFOR.     

Detection of Brucella , abortus , in the cervical mucus of nulliparous heifers

Sixteen nulliparous Holstein heifers were exposed artificially to Brucella , abortus , biotype 1 strain 2308. Attempts were made to recover the organism from blood, udder secretions and cervical mucus. Blood cultures 2 to 4 wk postexposure were positive. B . abortus , was recovered from one or more udder quarters in 11 heifers. B . abortus , was recovered from cervical mucus of one heifer on Day 18 postexposure. All heifers were serologically positive within 5 wk. The presence of B . abortus , in the nongravid uterus is transitory and associated with the bacteremic phase. It is limited or prevented in most heifers due to the effect of estrus. Nulliparous heifers are suitable candidates for use as donors of Brucella -free embryos, even where infection is known to exist in the herd.     

Effects of embryo-freezing and thawing techniques on the survivability of Brucella abortus

A suspension of a pathogenic strain (2308) of Brucella abortus was aliquoted, centrifuged, resuspended in 6 treatment media and quantitated. Ten 1-ml samples of each treatment were subjected to a standard embryo-freezing technique. The treatments were selected to examine the effects of 1) freezing and thawing, 2) cryoprotectants and 3) antibiotics on the survivability of Brucella suspended in embryo-support media. Five samples of each treatment were thawed and quantitated after a 2-wk storage period and five samples were thawed and quantitated after a 6-mo storage period. Means and percent reductions were determined for each treatment. There was no statistical difference between means at 2 wk and 6 mo within any treatment. Freezing and thawing caused a 64% reduction in the number of viable Brucella . The addition of antibiotics caused a 99.9% reduction in viability of the organism. Glycerol protected the organism during freezing and thawing in the absence of antibiotics but did not interfere with the high percent reduction seen when antibiotics were present. Dimethylsulfoxide (DMSO), however, not only protected the organism during freezing and thawing but also appeared to negate the effects of the antibiotics.     

Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins

Neisseria meningitidis FAM20 has recently been shown to produce two Fe-regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine-amino-acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (0RF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300 bp upstream of ORF1, and it contained a Fur protein-binding sequence found in the promoters of Fe-regulated Escherichia coli genes. DNA upstream of the ORF 1/frpC promoter was homologous to IStO76-like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC-like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.     

Preferential uptake of restriction fragments from a gonococcal cryptic plasmid by competent Neisseria gonorrhoeae

Factors involved in the specificity of DNA uptake by competent Neisseria gonorrhoeae were examined. Host-controlled modification did not affect uptake. Certain restriction fragments of the 4.2 kb gonococcal cryptic plasmid pFA1 and of the replicative form of the bacteriophage M13 were taken up in preference to others, independent of differences in fragment size. A 600 bp fragment from the 4.2 kb plasmid was cloned into pLES2, a gonococcal-Escherichia coli shuttle vector; the 600 bp fragment was taken up into a DNAase-I-resistant state in preference to the vector fragment. A second 370 bp fragment in pFA1 was also taken up preferentially. The 600 bp and 370 bp fragments share a 10 bp sequence, which is found in pFA1 only on fragments that were taken up readily. However, a fragment from M13 which was efficiently taken up did not contain this 10 bp sequence. In addition, this sequence was not sufficient to direct preferential DNA uptake by gonococci, since a recombinant plasmid containing this 10 bp sequence was not taken up appreciably better than the vector plasmid or another recombinant plasmid containing an unrelated 10 bp sequence. Sequence comparisons of the three restriction fragments which were preferentially taken up did not yield any consensus sequences greater than 7 bp. Although it is likely that efficient uptake of DNA by gonococci is determined by DNA structure, a single short sequence could not be found that accounted for specific uptake.     

A metabolic and genomic assessment of sugar fermentation profiles of the thermophilic Thermotogales,Fervidobacterium pennivorans

A metabolic, genomic and proteomic assessment of Fervidobacterium pennivorans strains was undertaken to clarify the metabolic and genetic capabilities of this Thermotogales species. The type strain Ven5 originally isolated from a hot mud spa in Italy, and a newly isolated strain (DYC) from a hot spring at Ngatamariki, New Zealand, were compared for metabolic and genomic differences. The fermentation profiles of both strains on cellobiose generated similar major end products (acetate, alanine, glutamate, H₂, and CO₂). The vast majority of end products produced were redox neutral, and carbon balances were in the range of 95–115%. Each strain showed distinct fermentation profiles on sugar substrates. The genome of strain DYC was sequenced and shown to have high sequence similarity and synteny with F. pennivorans Ven5 genome, suggesting they are the same species. The unique genome regions in Ven5, corresponded to genes involved in the Entner–Doudoroff pathway confirming our observation of DYC’s inability to utilize gluconate. Genome analysis was able to elucidate pathways involved in production of the observed end-products with the exception of alanine and glutamate synthesis which were resolved with less clarity due to poor sequence identity and missing critical enzymes within the pathway, respectively.

     

Inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae.

The genetics of low-level resistance to penicillin and other antibiotics in a clinical isolate and a multistep laboratory mutant of Neisseria gonorrhoea was studied by transformation. Mutations at three loci affected sensitivity to penicillin. Mutation at penA resulted in an eightfold increase in resistance to penicillin without affecting response to other antimicrobial agents. Mutation at ery resulted in a two- to fourfold increase in resistance to penicillin and similar increases in resistance to many other antibiotics, dyes, and detergents. Mutation at penB resulted in a fourfold increase in resistance to penicillin and tetracycline, the phenotypic expression of which was dependent on the presence of mutation at ery. The cumulative effect of mutations at penA, ery, and penB was an approximate 128-fold increase in penicillin resistance, to a minimum inhibitory concentration of 1.0 mug/ml. Low-level resistance to tetracycline or chloramphenicol was due to similar additive effects between mutations at the nonspecific ery and penB loci and a locus specific for resistance to each drug (tet and chl, respectively). No evidence was found for penicillinases or other drug-inactivating enzymes.     

Ribosomal Resistance to Streptomycin and Spectinomycin in Neisseria gonorrhoeae

A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae. The 70S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30S ribosomal subunit by studying antibiotic sensitivities of hybrid 70S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes.