Clostridium ljungdahlii is an anaerobic homoacetogen, able to ferment sugars, other organic compounds, or CO₂/H₂ and synthesis gas (CO/H₂). The latter feature makes it an interesting microbe for the ...biotech industry, as important bulk chemicals and proteins can be produced at the expense of CO₂, thus combining industrial needs with sustained reduction of CO and CO₂ in the atmosphere. Sequencing the complete genome of C. ljungdahlii revealed that it comprises 4,630,065 bp and is one of the largest clostridial genomes known to date. Experimental data and in silico comparisons revealed a third mode of anaerobic homoacetogenic metabolism. Unlike other organisms such as Moorella thermoacetica or Acetobacterium woodii, neither cytochromes nor sodium ions are involved in energy generation. Instead, an Rnf system is present, by which proton translocation can be performed. An electroporation procedure has been developed to transform the organism with plasmids bearing heterologous genes for butanol production. Successful expression of these genes could be demonstrated, leading to formation of the biofuel. Thus, C. ljungdahlii can be used as a unique microbial production platform based on synthesis gas and carbon dioxide/hydrogen mixtures.
Phenotypic and phylogenetic studies were performed on new isolates of a novel Gram-stain-positive, anaerobic, non-sporulating, rod-shaped bacterium isolated from a thermophilic biogas plant. The ...novel organisms were able to degrade crystalline cellulose. 16S rRNA gene comparative sequence analysis demonstrated that the isolates formed a hitherto unknown subline within the family Lachnospiraceae. As a representative of the whole group of isolates, strain T3/55T was further characterized. The closest relative of T3/55T among the taxa with validly published names is Mobilitalea sibirica, sharing 93.9% 16S rRNA gene sequence similarity. Strain T3/55T was catalase-negative, indole-negative, and produced acetate, ethanol and propionic acid as major end products from cellulose metabolism. The major cellular fatty acids (>1%) were 16 : 0 dimethyl acetal, 16 : 0 fatty acid methyl ester and 16 : 0 aldehyde. The DNA G+C content was 36.6 mol%. A novel genus and species, Herbinix hemicellulosilytica gen. nov., sp. nov., is proposed based on phylogenetic analysis and physiological properties of the novel isolate. Strain T3/55T ( = DSM 29228T = CECT 8801T), represents the type strain of Herbinix hemicellulosilytica gen. nov., sp. nov.
Acetic acid bacteria (AAB) are a group of Gram-negative, strictly aerobic bacteria, including 19 reported genera until 2021, which are widely found on the surface of flowers and fruits, or in ...traditionally fermented products. Many AAB strains have the great abilities to incompletely oxidize a large variety of carbohydrates, alcohols and related compounds to the corresponding products mainly including acetic acid, gluconic acid, gulonic acid, galactonic acid, sorbose, dihydroxyacetone and miglitol
the membrane-binding dehydrogenases, which is termed as AAB oxidative fermentation (AOF). Up to now, at least 86 AOF products have been reported in the literatures, but no any monograph or review of them has been published. In this review, at first, we briefly introduce the classification progress of AAB due to the rapid changes of AAB classification in recent years, then systematically describe the enzymes involved in AOF and classify the AOF products. Finally, we summarize the application of molecular biology technologies in AOF researches.
Clostridium acetobutylicum is a strict anaerobic organism that is used for biotechnological butanol fermentation. It ferments various hexoses and pentoses to solvents but prefers glucose presumably ...using a catabolite repression mechanism. Accordingly during growth on a mixture of
d-glucose and
d-xylose a typical diauxic growth pattern was observed. We used DNA microarrays and real-time RT-PCR to study gene expression during growth on
d-glucose,
d-xylose mixtures on a defined minimal medium together with monitoring substrate consumption and product formation. We identified two putative operons involved in
d-xylose degradation. The first operon (CAC1344–CAC1349) includes a transporter, a xylulose-kinase, a transaldolase, a transketolase, an aldose-1-epimerase and a putative xylose isomerase that has been annotated as an arabinose isomerase. This operon is induced by
d-xylose but was catabolite repressed by
d-glucose. A second operon (CAC2610–CAC2612) consists of a xylulose-kinase, a hypothetical protein and a gene that has been annotated as a
l-fucose isomerase that might in fact code for a xylose isomerase. This operon was induced by
d-xylose but was not subject to catabolite repression. In accordance with these results we identified a CRE site in the catabolite repressed operon but not in the operon that was not subject to catabolite repression.
Acetic acid bacteria (AAB) are Gram-negative obligate aerobics in Acetobacteraceae family. Producing acetic acid and brewing vinegars are one of the most important industrial applications of AAB, ...attributed to their outstanding ability to tolerate the corresponding stresses. Several unique acid resistance (AR) mechanisms in AAB have been revealed previously. However, their overall AR strategies are still less-comprehensively clarified. Consequently, omics analysis was widely performed for a better understanding of this field. Among them, transcriptome has recently obtained more and more attention. However, most currently reported transcriptomic studies were conducted under lab conditions and even in low-acidity environment, which may be unable to completely reflect the conditions that AAB confront under industrialized vinegar-brewing processes. In this study, we performed an RNA-Seq transcriptomic analysis concerning AAB’s AR mechanisms during a continuous and periodical industrial submerged vinegar fermentation process, where a single AAB strain performed the fermentation and the acetic acid concentration fluctuated between ~8% and ~12%, the highest acidity as far we know for transcriptomic studies. Samples were directly taken from the initial (CK), mid, and final stages of the same period of the on-going fermentation. 16S rRNA sequence analysis indicated the participation of
Komagataeibacter europaeus
in the fermentation. Transcriptomic results demonstrated that more genes were downregulated than upregulated at both mid and final stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrich analysis reflected that the upregulated genes mainly carried out tricarboxylic acid cycle and oxidative phosphorylation processes, probably implying a considerable role of acetic acid overoxidation in AR during fermentation. Besides, upregulation of riboflavin biosynthesis pathway and two NAD
+
-dependent succinate-semialdehyde dehydrogenase-coding genes suggested a critical role of succinate oxidation in AR. Meanwhile, downregulated genes were mainly ribosomal protein-coding ones, reflecting that the adverse impact on ribosomes initiates at the transcription level. However, it is ambiguous whether the downregulation is good for stress responding or it actually reflects the stress. Furthermore, we also assumed that the fermentation stages may have a greater effect on gene expression than acidity. Additionally, it is possible that some physiological alterations would affect the AR to a larger extent than changes in gene expression, which suggests the combination of molecular biology and physiology research will provide deeper insight into the AR mechanisms in AAB.
A new strain of xanthan-degrading bacteria identified as
sp. has been isolated from a xanthan thickener for food production. The strain was able to utilize xanthan as the only carbon source and to ...reduce the viscosity of xanthan-containing medium during cultivation. Comparative analysis of the secretomes of
sp. after growth on different media led to the identification of a xanthanase designated as
Xan9, which was isolated after recombinant production in
.
Xan9 could efficiently degrade the β-1,4-glucan backbone of xanthan after previous removal of pyruvylated mannose residues from the ends of the native xanthan side chains by xanthan lyase treatment (XLT-xanthan). Compared with xanthanase from
, xanthanase
Xan9 had a different module composition at the N- and C-terminal ends. The main putative oligosaccharides released from XLT-xanthan by
Xan9 cleavage were tetrasaccharides and octasaccharides. To explore the functions of the N- and C-terminal regions of the enzyme, truncated variants lacking some of the non-catalytic modules (
Xan9-C,
Xan9-N,
Xan9-C-N) were produced. Enzyme assays with the purified deletion derivatives, which all contained the catalytic glycoside hydrolase family 9 (GH9) module, demonstrated substantially reduced specific activity on XLT-xanthan of
Xan9-C-N compared with full-length
Xan9. The C-terminal module of
Xan9 was found to represent a novel carbohydrate-binding module of family CBM66 with binding affinity for XLT-xanthan, as was shown by native affinity polyacrylamide gel electrophoresis in the presence of various polysaccharides. The only previously known binding function of a CBM66 member is exo-type binding to the non-reducing fructose ends of the β-fructan polysaccharides inulin and levan.
Gluconobacter oxydans
, like all acetic acid bacteria, has several membrane-bound dehydrogenases, which oxidize a multitude of alcohols and polyols in a stereo- and regio-selective manner. Many ...membrane-bound dehydrogenases have been purified from various acetic acid bacteria, but in most cases without reporting associated sequence information. We constructed clean deletions of all membrane-bound dehydrogenases in
G
.
oxydans
621H and investigated the resulting changes in carbon utilization and physiology of the organism during growth on fructose, mannitol, and glucose. Furthermore, we studied the substrate oxidation spectra of a set of strains where the membrane-bound dehydrogenases were consecutively deleted using a newly developed whole-cell 2,6-dichlorophenolindophenol (DCPIP) activity assay in microtiter plates. This allowed a detailed and comprehensive in vivo characterization of each membrane-bound dehydrogenase in terms of substrate specificity. The assays revealed that general rules can be established for some of the enzymes and extended the known substrate spectra of some enzymes. It was also possible to assign proteins whose purification and characterization had been reported previously, to their corresponding genes. Our data demonstrate that there are less membrane-bound dehydrogenases in
G
.
oxydans
621H than expected and that the deletion of all of them is not lethal for the organism.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
Sourdough has played a significant role in human nutrition and culture for thousands of years and is still of eminent importance for human diet and the bakery industry. Lactobacillus sanfranciscensis ...is the predominant key bacterium in traditionally fermented sourdoughs.The genome of L. sanfranciscensis TMW 1.1304 isolated from an industrial sourdough fermentation was sequenced with a combined Sanger/454-pyrosequencing approach followed by gap closing by walking on fosmids. The sequencing data revealed a circular chromosomal sequence of 1,298,316 bp and two additional plasmids, pLS1 and pLS2, with sizes of 58,739 bp and 18,715 bp, which are predicted to encode 1,437, 63 and 19 orfs, respectively. The overall GC content of the chromosome is 34.71%. Several specific features appear to contribute to the ability of L. sanfranciscensis to outcompete other bacteria in the fermentation. L. sanfranciscensis contains the smallest genome within the lactobacilli and the highest density of ribosomal RNA operons per Mbp genome among all known genomes of free-living bacteria, which is important for the rapid growth characteristics of the organism. A high frequency of gene inactivation and elimination indicates a process of reductive evolution. The biosynthetic capacity for amino acids scarcely availably in cereals and exopolysaccharides reveal the molecular basis for an autochtonous sourdough organism with potential for further exploitation in functional foods. The presence of two CRISPR/cas loci versus a high number of transposable elements suggests recalcitrance to gene intrusion and high intrinsic genome plasticity.
A novel Gram-stain-positive, rod-shaped, anaerobic, thermophilic bacterium, strain GGR1T, was isolated from a thermophilic lab-scale biogas fermenter. The novel organism was effectively degrading ...crystalline cellulose. It seems to play a role in remineralization of plant biomass by hydrolysing its polysaccharides. 16S rRNA gene comparative sequence analysis demonstrated that the isolate formed a hitherto unknown subline within the family Ruminococcaceae. The closest phylogenetic relative of GGR1T among the taxa with validly published names was Clostridiumthermocellum, sharing 94.3 % 16S rRNA gene sequence similarity. Strain GGR1T was catalase-negative, indole-negative and produced acetate and ethanol as major end-products during fermentative cellulose utilization. The major cellular fatty acids (>1 %) were 16 : 0 iso fatty acid and 16 : 0 fatty acid. Cells were rod shaped and grew optimally at 60 °C and pH 7.0. The DNA G+C content was 34.9 mol%. A novel genus and species, Herbivoraxsaccincola gen. nov., sp. nov., is proposed on the basis of phylogenetic analysis and physiological properties of the novel isolate. Strain GGR1T (=DSM 101079T=CECT 9155T) represents the type strain for the novel genus and novel species Herbivoraxsaccincola gen. nov., sp. nov.
For the detailed molecular analysis, genomic modification, and application of acetic acid bacteria such as
Gluconobacter
in biotechnological processes, a simple markerless deletion system is ...essential. The available methods have either low efficiencies or their applicability is restricted to strains containing an
upp
mutation. We now developed a method based on counterselection by cytosine deaminase, encoded by the
codA
gene from
Escherichia coli
, in the presence of the fluorinated pyrimidine analogue 5-fluorocytosine (FC). The
codA
-encoded enzyme converts nontoxic FC to toxic 5-fluorouracil, which is channeled into the metabolism by the uracil phosphoribosyltransferase, encoded by the chromosomal
upp
gene of
Gluconobacter
. We found that the presence of
E
.
coli codB
, encoding a cytosine permease, was needed for a high efficiency of gene deletion. The system is applicable in wild-type strains because no preceding deletions are required. Based on the fact that a
codA
gene is absent and an
upp
gene is present in almost all acetic acid bacteria sequenced so far, the method should also be applicable for other genera of the
Acetobacteraceae
.
Celotno besedilo
Dostopno za:
CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
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