Glutamate residues at positions 162nd and 164th influence the beta-lactamase activity of SHV-14 obtained from Klebsiella pneumoniae

Abstract

Extensive production of SHV-14 beta-lactamase makes Klebsiella pneumoniae resistant to beta-lactams. The presence of omega-loop has been reported to influence the beta-lactamase activity, which is also present in SHV-14. Its omega-loop has three glutamates in nearly alternating positions, 162, 164 and 167 but their concise role on the behaviour of SHV-14 is unknown. To uncover the influence of each glutamate on SHV-14, we replaced glutamates with alanine and estimated the effect of each mutation by assessing the change in beta-lactam sensitivities in the surrogate Escherichia coli cells and catalytic efficiencies for hydrolysis with the purified proteins. Upon expression, the clone of wild-type SHV-14 aggravated the resistance of host by 60–500 folds against penicillin and cephalosporin groups of antibiotics. However, the expression of mutated enzymes (especially E164A) substantially reduced the resistance level as compared to the wild-type, and the results were in synchrony with the estimated enzymatic efficiencies of wild-type and mutated proteins. Therefore, with further support from the in silico analysis, we hypothesize that mutation at the glutamate residues in the omega-loop of SHV-14 can considerably modulate the beta-lactam sensitivity and hydrolysis; thus, revealing the importance of such glutamates as the target for inhibitor design in future.

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Microbial carbon dioxide fixation: new tricks for an old game

Abstract

The exploitation of petroleum as energy and material source opened unprecedented possibilities for the development of our human societies, but only now we realize that the use of fossil resources comes at devastatingly high environmental costs. Consequently, our efforts to tap other carbon sources are steadily increasing. Industrial microbiology has the potential to use carbon dioxide directly as carbon source, thereby converting a foe into a friend. This thematic issue of FEMS Microbiology Letters sheds some light on recent developments for the understanding of microbial pathways for carbon dioxide fixation and on strategies for their industrial exploitation.

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Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria

Abstract

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rdv. sulfidophilum is a non-sulfur phototrophic marine bacterium, which is known to form flocs, structured communities of cells, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic will also be described.

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Rapid screening method for detecting highly pathogenic Streptococcus intermedius strains carrying a mutation in the lacR gene.

Abstract

Streptococcus intermedius is a member of the normal human commensal flora and secretes a human-specific cytolysin intermedilysin (ILY) as a major virulence factor. Expression of ily is repressed by LacR and loss-of-function mutations of LacR are observed in many ILY high-producing strains isolated from deep-seated abscesses, suggesting that high ILY production is necessary for increased virulence. However, because ILY exhibits no β-hemolysis on animal blood agar plates, differentiating ILY high- and low-producing strains using conventional laboratory methods is not possible. Interestingly, S. intermedius also produces glycosidases, including MsgA and NanA, which exhibit N-acetyl-β-d-glucosaminidase and neuraminidase activities, respectively. Moreover, MsgA expression, but not NanA, is negatively regulated by LacR. Here we measured the activities of MsgA, NanA and ILY in strains isolated from clinical specimens and dental plaque to determine the correlation between these glycosidase activities and ILY hemolytic activity. Hemolytic activity showed a strong positive correlation with MsgA and a weak negative correlation with NanA activities. Therefore, we calculated the ratio of MsgA and NanA activity (M/N ratio). This value showed a stronger positive correlation (r = 0.81) with ILY hemolytic activity and many strains with high M/N ratios ( > 2) were ILY high-producers with loss-of-function mutations in LacR.

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The impact of environmental factors on CO2 fixation by microalgae

Abstract

Microalgae are among the most productive biological systems for converting sunlight into chemical energy which is used to capture and transform inorganic carbon into biomass. The efficiency of CO₂ capture depends on the cultivation system configuration (photobioreactors or open systems) and can vary according to the state of the algal physiology, the chemical composition of the nutrient medium and environmental factors, such as irradiance, temperature and pH. This mini-review is focused on some of the most important environmental factors determining photosynthetic activity, CO₂ biofixation, cell growth rates and biomass productivities by microalgae. These include CO₂ and O₂ concentrations, light intensity, cultivation temperature, nutrients, etc. Finally, a review on operation of microalgae cultivation systems in outdoors is presented as an example of the impact of environmental conditions on biomass productivity and CO₂ fixation.

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Poly(3-hydroxybutyrate) hyperproduction by a global nitrogen regulator NtrB mutant strain of Paracoccus denitrificans PD1222

Abstract

Paracoccus denitrificans PD1222 accumulates short-length polyhydroxyalkanoates, poly(3-hydroxybutyrate), under nitrogen deficient conditions. Polyhydroxybutyrate metabolism requires the 3-ketoacyl-CoA thiolase PhaA, the acetoacetyl-CoA dehydrogenase/reductase PhaB, and the synthase PhaC for polymerization. Additionally, P. denitrificans PD1222 grows aerobically with nitrate as sole nitrogen source. Nitrate assimilation is controlled negatively by ammonium through the two-component NtrBC system. NtrB is a sensor kinase that autophosphorylates a histidine residue under low nitrogen concentrations and, in turn transfers a phosphoryl group to an aspartate residue of the response regulator NtrC protein, which acts as a transcriptional activator of the P. denitrificans PD1222 nasABGHC genes. The P. denitrificans PD1222 NtrB mutant was unable to use nitrate efficiently as nitrogen source when compared to the wild-type strain, and it also overproduced poly(3-hydroxybutyrate). Acetyl-CoA concentration in the P. denitrificans PD1222 NtrB mutant strain was higher than in the wild-type strain. The expression of the phaC gene was also increased in the NtrB mutant when compared to the wild-type strain. These results suggest that accumulation of poly(3-hydroxybutyrate) in the NtrB mutant strain of PD1222 responds to the high levels of acetyl-CoA that accumulate in the cytoplasm as consequence of its inability to efficiently use nitrate as nitrogen source.

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Relationship between Escherichia coli growth rate and bacterial susceptibility to ciprofloxacin

Abstract

The effect of Escherichia coli growth rate on its susceptibility to ciprofloxacin was investigated using bacteria grown on different carbon sources and harboring mutations in genes encoding tricarboxylic acid cycle enzymes. A 1-h treatment of the wild type (wt) grown on glucose, succinate, malate, α-ketoglutarate or acetate with 0.3 μg ml^–1 ciprofloxacin decreased the number of surviving cells (CFU ml^–1), 560, 110, 74, 62 and 5 times, respectively. Among the mutants tested, sucB strain, which grew 1.75 times slower than wt, was 7.4-fold more tolerant to 0.3 μg ml^–1 of ciprofloxacin than wt. Strong inverse correlations between log(CFU ml^–1) after 1-h exposure to 0.3 and 3.0 μg ml^–1 ciprofloxacin and the specific growth rate prior to antibiotic treatment (r = − 0.93 and − 0.96, respectively) were observed. Data from the current and previous studies on the inhibitory effect of ciprofloxacin on cultures exhibiting a wide range of growth rates (0.01–1.3 h^–1) were collated. Statistical analysis revealed a significant inverse correlation between log(CFU ml^–1) after exposure to 3.0 μg ml^–1 of ciprofloxacin and the specific bacterial growth rate prior to antibiotic exposure (r = − 0.92). These data may be used in a design of antibiotic treatment protocols.

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