Anaerobic Fermentative Growth Research Articles

L-arginine supplementation abrogates hypoxia-induced virulence of Staphylococcus aureus in a murine diabetic pressure wound model.

Diabetic foot ulcers (DFUs) are the most common complications of diabetes resulting from hyperglycemia leading to ischemic hypoxic tissue and nerve damage. Staphylococcus aureus is the most frequently isolated bacteria from DFUs and causes severe necrotic infections leading to amputations with a poor 5-year survival rate. However, very little is known about the mechanisms by which S. aureus dominantly colonizes and causes severe disease in DFUs. Herein, we utilized a pressure wound model in diabetic TALLYHO/JngJ mice to reproduce ischemic hypoxic tissue damage seen in DFUs and demonstrated that anaerobic fermentative growth of S. aureus significantly increased the virulence and the severity of disease by activating two-component regulatory systems leading to expression of virulence factors. Our in vitro studies showed that supplementation of nitrate as a terminal electron acceptor promotes anaerobic respiration and suppresses the expression of S. aureus virulence factors through inactivation of two-component regulatory systems, suggesting potential therapeutic benefits by promoting anaerobic nitrate respiration. Our in vivo studies revealed that dietary supplementation of L-arginine (L-Arg) significantly attenuated the severity of disease caused by S. aureus in the pressure wound model by providing nitrate. Collectively, these findings highlight the importance of anaerobic fermentative growth in S. aureus pathogenesis and the potential of dietary L-Arg supplementation as a therapeutic to prevent severe S. aureus infection in DFUs.IMPORTANCES. aureus is the most common cause of infection in DFUs, often resulting in lower-extremity amputation with a distressingly poor 5-year survival rate. Treatment for S. aureus infections has largely remained unchanged for decades and involves tissue debridement with antibiotic therapy. With high levels of conservative treatment failure, recurrence of ulcers, and antibiotic resistance, a new approach is necessary to prevent lower-extremity amputations. Nutritional aspects of DFU treatment have largely been overlooked as there has been contradictory clinical trial evidence, but very few in vitro and in vivo modelings of nutritional treatment studies have been performed. Here we demonstrate that dietary supplementation of L-Arg in a diabetic mouse model significantly reduced duration and severity of disease caused by S. aureus. These findings suggest that L-Arg supplementation could be useful as a potential preventive measure against severe S. aureus infections in DFUs.

Environmental Triggers of lrgA Expression in Streptococcus mutans.

The cidAB and lrgAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans, including oxidative stress tolerance, biofilm formation, and autolysis. Here we investigate the regulation of lrgAB which in S. mutans shows a complex dependence on growth conditions that is not fully understood. By combining single-cell imaging of a fluorescent gene reporter with microfluidic control of the extracellular environment, we identify specific environmental cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. We find that the very abrupt activation of lrgA at stationary phase is tightly synchronized across the population. This activation is controlled by a small number of inputs that are sensitive to growth phase: extracellular pyruvate, glucose, and molecular oxygen. Activation of lrgA appears to be self-limiting, so that strong expression of lrgA is confined to a short interval of time. lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies of other bacteria showing that homologs of lrgAB participate, with input from lytST, in the reimport of pyruvate for anaerobic fermentative growth.

Genome-Wide Analysis of ResD, NsrR, and Fur Binding in Bacillus subtilis during Anaerobic Fermentative Growth by In Vivo Footprinting.

Upon oxygen limitation, the Bacillus subtilis ResE sensor kinase and its cognate ResD response regulator play primary roles in the transcriptional activation of genes functioning in anaerobic respiration. The nitric oxide (NO)-sensitive NsrR repressor controls transcription to support nitrate respiration. In addition, the ferric uptake repressor (Fur) can modulate transcription under anaerobic conditions. However, whether these controls are direct or indirect has been investigated only in a gene-specific manner. To gain a genomic view of anaerobic gene regulation, we determined the genome-wide in vivo DNA binding of ResD, NsrR, and Fur transcription factors (TFs) using in situ DNase I footprinting combined with chromatin affinity precipitation sequencing (ChAP-seq; genome footprinting by high-throughput sequencing [GeF-seq]). A significant number of sites were targets of ResD and NsrR, and a majority of them were also bound by Fur. The binding of multiple TFs to overlapping targets affected each individual TF's binding, which led to combinatorial transcriptional control. ResD bound to both the promoters and the coding regions of genes under its positive control. Other genes showing enrichment of ResD at only the promoter regions are targets of direct ResD-dependent repression or antirepression. The results support previous findings of ResD as an RNA polymerase (RNAP)-binding protein and indicated that ResD can associate with the transcription elongation complex. The data set allowed us to reexamine consensus sequence motifs of Fur, ResD, and NsrR and uncovered evidence that multiple TGW (where W is A or T) sequences surrounded by an A- and T-rich sequence are often found at sites where all three TFs competitively bind.IMPORTANCE Bacteria encounter oxygen fluctuation in their natural environment as well as in host organisms. Hence, understanding how bacteria respond to oxygen limitation will impact environmental and human health. ResD, NsrR, and Fur control transcription under anaerobic conditions. This work using in situ DNase I footprinting uncovered the genome-wide binding profile of the three transcription factors (TFs). Binding of the TFs is often competitive or cooperative depending on the promoters and the presence of other TFs, indicating that transcriptional regulation by multiple TFs is much more complex than we originally thought. The results from this study provide a more complete picture of anaerobic gene regulation governed by ResD, NsrR, and Fur and contribute to our further understanding of anaerobic physiology.

Global Transcriptional Control by NsrR in Bacillus subtilis

The NO-sensitive NsrR repressor of Bacillus subtilis, which carries a [4Fe-4S] cluster, controls transcription of nasD and hmp (class I regulation) under anaerobic conditions. Here, we describe another class of NsrR regulation (class II regulation) that controls a more diverse collection of genes. Base substitution analysis showed that [4Fe-4S]-NsrR recognizes a partial dyad symmetry within the class I cis-acting sites, whereas NO-insensitive interaction of NsrR with an A+T-rich class II regulatory site showed relaxed sequence specificity. Genome-wide transcriptome studies identified genes that are under the control of the class II NsrR regulation. The class II NsrR regulon includes genes controlled by both AbrB and Rok repressors, which also recognize A+T-rich sequences, and by the Fur repressor. Transcription of class II genes was elevated in an nsrR mutant during anaerobic fermentative growth with pyruvate. Although NsrR binding to the class II regulatory sites was NO insensitive in vitro, transcription of class II genes was moderately induced by NO, which involved reversal of NsrR-dependent repression, suggesting that class II repression is also NO sensitive. In all NsrR-repressed genes tested, the loss of NsrR repressor activity was not sufficient to induce transcription as induction required the ResD response regulator. The ResD-ResE signal transduction system is essential for activation of genes involved in aerobic and anaerobic respiration. This study indicated coordinated regulation between ResD and NsrR and uncovered a new role of ResD and NsrR in transcriptional regulation during anaerobiosis of B. subtilis.

The Transcription Factor AlsR Binds and Regulates the Promoter of the alsSD Operon Responsible for Acetoin Formation in Bacillus subtilis

Bacillus subtilis forms acetoin under anaerobic fermentative growth conditions and as a product of the aerobic carbon overflow metabolism. Acetoin formation from pyruvate requires α-acetolactate synthase and acetolactate decarboxylase, both encoded by the alsSD operon. The alsR gene, encoding the LysR-type transcriptional regulator AlsR, was found to be essential for the in vivo expression of alsSD in response to anaerobic acetate accumulation, the addition of acetate, low pH, and the aerobic stationary phase. The expressions of the alsSD operon and the alsR regulatory gene were independent of other regulators of the anaerobic regulatory network, including ResDE, Fnr, and ArfM. A negative autoregulation of alsR was observed. In vitro transcription from the alsSD promoter using purified B. subtilis RNA polymerase required AlsR. DNA binding studies with purified recombinant AlsR in combination with promoter mutagenesis experiments identified a 19-bp high-affinity palindromic binding site (TAAT-N(11)-ATTA) at positions -76 to -58 (regulatory binding site [RBS]) and a low-affinity site (AT-N(11)-AT) at positions -41 to -27 (activator binding site [ABS]) upstream of the transcriptional start site of alsSD. The RBS and ABS were found to be essential for in vivo alsSD transcription. AlsR binding to both sites induced the formation of higher-order, transcription-competent complexes. The AlsR protein carrying the S100A substitution at the potential coinducer binding site still bound to the RBS and ABS. However, AlsR(S100A) failed to form the higher-order complex and to initiate in vivo and in vitro transcription. A model for AlsR promoter binding and transcriptional activation was deduced.

Iron restriction induces preferential down-regulation of H2-consuming over H2-evolving reactions during fermentative growth of Escherichia coli

BackgroundEscherichia coli synthesizes three anaerobically inducible [NiFe]-hydrogenases (Hyd). All three enzymes have a [NiFe]-cofactor in the large subunit and each enzyme also has an iron-sulfur-containing small subunit that is required for electron transfer. In order to synthesize functionally active Hyd enzymes iron must be supplied to the maturation pathways for both the large and small subunits. The focus of this study was the analysis of the iron uptake systems required for synthesis of active Hyd-1, Hyd-2 and Hyd-3 during fermentative growth.ResultsA transposon-insertion mutant impaired in hydrogenase enzyme activity was isolated. The mutation was in the feoB gene encoding the ferrous iron transport system. The levels of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 as determined by specific in-gel activity staining were reduced at least 10-fold in the mutant after anaerobic fermentative growth in minimal medium, while the hydrogen-evolving Hyd-3 activity was less severely affected. Supplementation of the growth medium with ferric iron, which is taken up by e.g. the siderophore enterobactin, resulted in phenotypic complementation of the feoB mutant. Growth in rich medium demonstrated that a mutant lacking both the ferrous iron transport system and enterobactin biosynthesis (entC) was devoid of Hyd-1 and Hyd-2 activity but retained some hydrogen-evolving Hyd-3 activity. Analysis of crude extracts derived from the feoB entC double null mutant revealed that the large subunits of the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 were absent. Analysis of lacZ fusions demonstrated, however, that expression of the hya, hyb and hyc operons was reduced only by maximally 50% in the mutants compared with the wild type.ConclusionsOur findings demonstrate that the ferrous iron transport system is the principal route of iron uptake for anaerobic hydrogenase biosynthesis, with a contribution from the ferric-enterobactin system. Hydrogen-oxidizing enzyme function was abolished in a feoB entC double mutant and this appears to be due to post-translational effects. The retention of residual hydrogen-evolving activity, even in the feoB entC double null mutant suggests that sufficient iron can be scavenged to synthesize this key fermentative enzyme complex in preference to the hydrogen-uptake enzymes.

Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain

The Gram-positive bacterium Bacillus cereus is a facultative anaerobe that is still poorly characterized metabolically. In this study, the aerobic vegetative growth and anaerobic vegetative growth of the food-borne pathogen B. cereus F4430/73 strain were compared with those of the genome-sequenced ATCC14579 strain using glucose and glycerol as fermentative and nonfermentative carbon sources, respectively. Uncontrolled batch cultures on several defined media showed that B. cereus strains had high amino acid or pyruvate requirements for anaerobic fermentative growth. In addition, growth performance was considerably improved by maintaining the pH of the culture medium near neutrality. Spectra of fermentation by-products were typically (per mole of glucose) 0.2-0.4 acetate, 1.1-1.4 L-lactate, 0.3-0.4 formate, and 0.05-0.2 ethanol with only traces of succinate, pyruvate, and 2,3-butanediol. These spectra were drastically changed in the presence of 20 mmol nitrate x L(-1), which stimulated anaerobic growth. During anaerobic and aerobic respiration, the persistent production of acetate and other by-products indicated overflow metabolisms. This was especially true in glucose-grown cells for which respiratory complex III made only a minor contribution to growth. Surprisingly, oxygen uptake rates linked to the cytochrome c and quinol branches of the respiratory chain were maintained at high levels in anaerobic, respiring, or fermenting cells. Growth and metabolic features of B. cereus F4430/73 are discussed using biochemical and genomic data.

Pseudovibrio denitrificans gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification

Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34(T) and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan. They were Gram-negative. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed. They were motile by means of one to several lateral or subpolar flagella. Both strains required NaCl for growth and exhibited optimal growth at about 30 degrees C, pH 8 and 3 % NaCl. They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates. Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate. The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0. DN34(T) and DN33 had DNA G+C contents of 51.7 and 51.6 mol%, respectively. Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen. nov. is proposed. Pseudovibrio denitrificans sp. nov. is the type species, with DN34(T) (=BCRC 17323(T)=JCM 12308(T)) as the type strain.

The Pseudomonas aeruginosa hemA promoter is regulated by Anr, Dnr, NarL and Integration Host Factor.

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.

Redox potential is a determinant in the Escherichia coli anaerobic fermentative growth and survival: effects of impermeable oxidant

Decrease of redox potential ( E h) down to −550–600 mV in the Escherichia coli culture is observed during growth in either anaerobic or aerobic conditions. The E. coli growth and survival under anaerobic fermentative conditions were found to be strongly inhibited by potassium ferricyanide in the concentration of 1 mM, when E h was decreased to −50–100 mV. This oxidant also resulted in approximately 2-fold decrease of total and N, N′-dicyclohexylcarbodiimide (DCCD)-inhibited H + efflux, 2.5-fold inhibition in K + influx, 1.5-fold less K + accumulation, and delayed a decrease in E h to negative values by bacteria. K 3[Fe(CN) 6] was shown to block an ATP-dependent increase in the amount of accessible thiol groups of membrane vesicles that was inhibited by DCCD, and this inhibition by the oxidant could be recovered by dithiothreitol. These effects were not observed with cells growing under aerobic conditions. The effects of K 3[Fe(CN) 6], an impermeable oxidant, might be explained by the fact that redox potential is a determinant in the E. coli anaerobic fermentative growth and survival that has a regulatory role in maintaining H + and K fluxes and the number of accessible thiol groups on membrane.

Alterococcus agarolyticus, gen.nov., sp.nov., a halophilic thermophilic bacterium capable of agar degradation

Five strains of facultatively anaerobic moderately thermophilic bacteria were isolated from two hot springs in the intertidal zone of Lutao, Taiwan. They produced extracellular agarase on agar medium, yielding reducing sugars and organic acids as the end products under either aerobic or anaerobic conditions. The growth temperature range was approximately 38–58°C with an optimal temperature of about 48°C. The five strains tolerated a relatively narrow pH range from 7.0 to 8.5. They were Gram-negative halophiles growing optimally at 2.0–2.5% NaCl (ca. 0.34–0.43 M). They were capable of anaerobic growth by fermenting glucose and producing various organic acids such as butyrate, propionate, formate, lactate, and acetate. Cells grown in liquid medium were motile monotrichous cocci, normally 0.8–0.9 µm in diameter. They possessed saturated anteiso-15-carbon acid (anteiso-C15:0) as the most abundant cellular fatty acid (46.0–51.3 mo1%) and had G+C contents ranging from 65.5 to 67.0 mo1%. They are the first thermophiles found to degrade agar and also the first halophilic thermophilic bacteria known to be capable of both aerobic and anaerobic fermentative growth. These bacteria are considered to represent a new genus that we named Alterococcus, and Alterococcus agarolyticus is the type species. Key words: Alterococcus agarolyticus, thermophilic bacteria, halophilic bacteria, agar-degrading bacteria, fermentative bacteria.

Alterococcus agarolyticus, gen.nov., sp.nov., a halophilic thermophilic bacterium capable of agar degradation.

Five strains of facultatively anaerobic moderately thermophilic bacteria were isolated from two hot springs in the intertidal zone of Lutao, Taiwan. They produced extracellular agarase on agar medium, yielding reducing sugars and organic acids as the end products under either aerobic or anaerobic conditions. The growth temperature range was approximately 38-58 degrees C with an optimal temperature of about 48 degrees C. The five strains tolerated a relatively narrow pH range from 7.0 to 8.5. They were Gram-negative halophiles growing optimally at 2.0-2.5% NaCl (ca. 0.34-0.43 M). They were capable of anaerobic growth by fermenting glucose and producing various organic acids such as butyrate, propionate, formate, lactate, and acetate. Cells grown in liquid medium were motile monotrichous cocci, normally 0.8-0.9 micron in diameter. They possessed saturated anteiso-15-carbon acid (anteiso-C15:0) as the most abundant cellular fatty acid (46.0-51.3 mo1%) and had G + C contents ranging from 65.5 to 67.0 mo1%. They are the first thermophiles found to degrade agar and also the first halophilic thermophilic bacteria known to be capable of both aerobic and anaerobic fermentative growth. These bacteria are considered to represent a new genus that we named Alterococcus, and Alterococcus agarolyticus is the type species.

Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth.

Bacillus subtilis can grow anaerobically by respiration with nitrate as a terminal electron acceptor. In the absence of external electron acceptors, it grows by fermentation. Identification of fermentation products by using in vivo nuclear magnetic resonance scans of whole cultures indicated that B. subtilis grows by mixed acid-butanediol fermentation but that no formate is produced. An ace mutant that lacks pyruvate dehydrogenase (PDH) activity was unable to grow anaerobically and produced hardly any fermentation product. These results suggest that PDH is involved in most or all acetyl coenzyme A production in B. subtilis under anaerobic conditions, unlike Escherichia coli, which uses pyruvate formate lyase. Nitrate respiration was previously shown to require the ResDE two-component signal transduction system and an anaerobic gene regulator, FNR. Also required are respiratory nitrate reductase, encoded by the narGHJI operon, and moaA, involved in biosynthesis of a molybdopterin cofactor of nitrate reductase. The resD and resDE mutations were shown to moderately affect fermentation, but nitrate reductase activity and fnr are dispensable for fermentative growth. A search for genes involved in fermentation indicated that ftsH is required, and is also needed to a lesser extent for nitrate respiration. These results show that nitrate respiration and fermentation of B. subtilis are governed by divergent regulatory pathways.