Emulsan production by Acinetobacter calcoaceticus in the presence of chloramphenicol.

When exponentially growing cultures of Acinetobacter calcoaceticus RAG-1 or RAG-92 were either treated with inhibitors of protein synthesis or starved for a required amino acid, there was a stimulation in the production of emulsan, an extracellular polyanionic emulsifier. Emulsan synthesis in the presence of chloramphenicol was dependent on utilizable sources of carbon and nitrogen and was inhibited by cyanide or azide or anaerobic conditions. Radioactive tracer experiments indicated that the enhanced production of emulsan after the addition of chloramphenicol was due to both the release of material synthesized before the addition of the antibiotic (40%) and de novo synthesis of the polymer (60%). Chemical analysis of RAG-1 cells demonstrated large amounts of polymeric amino sugars; it was estimated that cell-associated emulsan comprised about 15% of the dry weight of growing cells. The data are consistent with the hypothesis that a polymeric precursor of emulsan accumulates on the cell surface during the exponential growth phase; in the stationary phase or during inhibition of protein synthesis, the polymer is released as a potent emulsifier.     

Physiological studies on the oxidation of 3-chlorobenzoate by Acinetobacter calcoaceticus strain Bs 5

Summary Acinetobacter calcoaceticus, strain Bs 5, oxidized 3-chlorobenzoate only in the presence of a readily available energy source such as succinate or pyruvate. Cultures of the organism growing in the presence of 3-chlorobenzoate at concentrations greater than 250 M turned darkbrown on prolonged incubation. After centrifugation and acidificition of the supernatants to pH 2, a dark pigment could be precipitated. It was shown to contain bound chlorine and probably originated from 3-chlorocatechol and 4-chlorocatechol, which accumulated in cultures growing in the presence of 3-chlorobenzoate.3-Chlorobenzoate induced the ability to oxidize benzoate and catechol in the bacteria, the latter by orthofission. In the presence of an energy source, 3-chlorobenzoate-induced cells oxidized 3-chlorobenzoate at a rate, which was about 25% of the turnover rate for benzoate.In a continuous culture of the organism, growing in the presence of 1 mM 3-chlorobenzoate at a dilution rat of 0.025 h^–1, all culture parameters including the ability of the cells to oxidize benzoate, were oscillating. This oscillation was attributed to the transient build-up of toxic concentrations of chlorocatechols. 4-chlorocatechol was shown to be about six times more toxic than 3-chlorobenzoate. In another series of continuous culture experiments, 3-chlorobenzoate induced the synthesis of the benzoate oxidizing enzyme system of A. calcoaceticus strain Bs 5 at a concentration of 2.5 M, but appeared to lose its inducing potential towards this enzyme system at about 0.5 M.     

Mapping of the tryptophan genes of Acinetobacter calcoaceticus by transformation

Auxotrophs of Acinetobacter calcoaceticus blocked in each reaction of the synthetic pathway from chorismic acid to tryptophan were obtained after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. One novel class was found to be blocked in both anthranilate and p-aminobenzoate synthesis; these mutants (trpG) require p-aminobenzoate or folate as well as tryptophan (or anthranilate) for growth. The loci of six other auxotrophic classes requiring only tryptophan were defined by growth, accumulation, and enzymatic analysis where appropriate. The trp mutations map in three chromosomal locations. One group contains trpC and trpD (indoleglycerol phosphate synthetase and phosphoribosyl transferase) in addition to trpG mutations; this group is closely linked to a locus conferring a glutamate requirement. Another cluster contains trpA and trpB, coding for the two tryptophan synthetase (EC 4.2.1.20) subunits, along with trpF (phosphoribosylanthranilate isomerase); this group is weakly linked to a his marker. The trpE gene, coding for the large subunit of anthranilate synthetase, is unlinked to any of the above. This chromosomal distribution of the trp genes has not been observed in other organisms.     

Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans

Two types of Indian crude oil (Bombay High and Gujarat) were tested for their biodegradability by Acinetobacter calcoaceticus and Alcaligenes odorans. Acinetobacter calcoaceticus S30 and Alc. odorans P20 degraded Bombay High crude oil by 50% and 45%, while only 29% and 37% of Gujarat crude oil (heavy crude oil) was degraded by these isolates, respectively. Acinetobacter calcoaceticus and Alc. odorans in combination deraded 58% and 40% of Bombay High and Gujarat crude oils, respectively, which were significantly higher than that of by individual cultures. Acinetobacter calcoaceticus S30 degraded more of the alkanes fraction than the aromatics fraction of both crude oils. GC fingerprinting of alkane fraction showed major degradation of heptadecane (C₁₇), octadecane (C₁₈), nonadecane (C₁₉), eicosane (C₂₀), docosane (C₂₂), tricosane (C₂₃) and tetracosane (C₂₄) of crude oil, while the Alc. odorans P20 degraded alkanes and aromatics equally. The asphaltenic component increased in both types of crude oil after biodegradation. The two strains grew very well on n -alkane up to C₃₃ as well as on pristane (branched-chain alkane) but could not grow on cycloalkanes. Acinetobacter calcoaceticus S30 could not grow on pure polycyclic aromatic hydrocarbon (PAH) compounds except naphthalene but Alc. odorans P20 could grow on anthracene, phenanthrene, dibenzothiophene, fluorene, fluoranthene, pyrene and chrysene.     

Catalysis of the electrochemical reduction of oxygen by bacteria isolated from electro-active biofilms formed in seawater

Biofilms formed in aerobic seawater on stainless steel are known to be efficient catalysts of the electrochemical reduction of oxygen. Based on their genomic analysis, seven bacterial isolates were selected and a cyclic voltammetry (CV) procedure was implemented to check their electrocatalytic activity towards oxygen reduction. All isolates exhibited close catalytic characteristics. Comparison between CVs recorded with glassy carbon and pyrolytic graphite electrodes showed that the catalytic effect was not correlated with the surface area covered by the cells. The low catalytic effect obtained with filtered isolates indicated the involvement of released redox compounds, which was confirmed by CVs performed with adsorbed iron-porphyrin. None of the isolates were able to form electro-active biofilms under constant polarization. The capacity to catalyze oxygen reduction is shown to be a widespread property among bacteria, but the property detected by CV does not necessarily confer the ability to achieve stable oxygen reduction under constant polarization.     

Effects of carbon sources on extracellular lipase production and lipA transcription in Acinetobacter calcoaceticus

  The effects of lactic acid, oleic acid, gum arabic and their mutual interactions, on the production of extracellular lipase and the regulation of the expression of the lipase encoding gene (lipA) in Acinetobacter calcoaceticus were investigated. Formation of extracellular lipase was measured in culture supernatants of wild-type strain BD413 and expression of the lipA gene was monitored in vivo with a chromosomal fusion of lipA to lacZ. At the level of lipA expression only oleic acid had a significant effect; it lowered expression. Neither gum arabic nor lactic acid had any effect on lipA expression. On the other hand, the yield of extracellular lipase increased 2–5 times by the addition of gum arabic, possibly due to the release of cell surface-bound lipase. An interaction between oleic acid and lactic acid was also detected. Journal of Industrial Microbiology & Biotechnology (2000) 24, 25–30. Received 03 May 1999/ Accepted in revised form 04 September 1999     

Degradation of furazolidone by bacteria Acinetobacter calcoaceticus T32, Pseudomonas putida SP1 and Proteus mirabilis V7

Furazolidone (FZD) has been widely used as an antibacterial and antiprotozoal feed additive for poultry, cattle and farmed fish. Since FZD has been shown to have mutagenic, genotoxic and potentially carcinogenic properties when tested in a variety of systems, there is an increasing need to find a way to remove FZD from contaminated environments. In this report, three bacterial strains Acinetobacter calcoaceticus T32, Pseudomonas putida SP1 and Proteus mirabilis V7 capable of degrading FZD effectively were isolated, identified and characterized. The reduced FZD concentration after degradation was determined by HPLC. After bacterial cells were grown in the media containing 5 mg l⁻¹ FZD for 5 days, almost all FZD was degraded by A. calcoaceticus T32, and more than 50% of FZD was degraded by P. putida SP1 and P. mirabilis V7, respectively. Bacterial GST activity of A. calcoaceticus T32, P. putida SP1 and P. mirabilis V7 was determined to be influenced by different FZD concentrations. Cytotoxicity analysis showed that FZD was degraded to the metabolites with far less cytotoxicity compared to FZD. The inoculation of bacterial strains A. calcoaceticus T32, P. putida SP1 and P. mirabilis V7 into FZD-contained media resulted in a higher degradation efficiency than natural degradation, which indicated the potential application of these strains in treatment of FZD-polluted freshwater or seawater environments.     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

Microbial hydroxylation of indole to 7-hydroxyindole by Acinetobacter calcoaceticus strain 4-1-5

A screening study yielded Acinetobacter calcoaceticus strain 4-1-5, which is capable of hydroxylating indole to 7-hydroxyindole. Strain 4-1-5 grew on terephthalate as the sole source of carbon and energy and hydroxylated indole to 7-hydroxyindole by cometabolism of indole using terephthalate as cosubstrate. Strain 4-1-5 produced 0.574 mM of 7-hydroxyindole at 2.38 mM indole in 24 h with the cell growth.     

Recovery of DNA from the Acinetobacter calcoaceticus chromosome by gap repair.

A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.     

Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.     

An in vivo analysis of the energetics of aldose oxidation by Acinetobacter calcoaceticus

Summary A continuous culture study was made of the energetics of oxidation of various aldose sugars by Acinetobacter calcoaceticus LMD 79.41. The consumption of aldoses during carbon- and energy-limited growth of the organism on mixtures of acetate and an aldose was independent of the pH of the culture. Acid production, however, was strongly dependent on this parameter. It is shown that aldose consumption without concurrent acid production is due to formation of the corresponding lactone, the hydrolysis of which is pH-dependent. The cell yield of A. calcoaceticus on mixtures of acetate and glucose or xylose was much higher than during growth on acetate alone. This increase in cell yield was, however, dependent on the pH of the culture. Only at pH values which permitted a high rate of lactone hydrolysis an enhancement of the cell yield was observed. These results suggest that lactone hydrolysis has an important bearing on the efficiency of periplasmic oxidation of aldoses in bacteria.     

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds

Aims: The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds. Methods: Single species biofilms of a Pseudomonas aeruginosa or a methicillin‐resistant Staphylococcus aureus were grown in either colony biofilm or drip‐flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation. Conclusions: Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro‐organism in the same biofilm model. For each micro‐organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth‐2,3‐dimercaptopropanol (BisBAL) at 40–80 μg ml^?1 achieved >7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth‐1,3‐propanedithiol/bismuth‐2‐mercaptopyridine N‐oxide (BisBDT/PYR) achieved a 4·9 log reduction. Significance and Impact of the Study: Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.