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Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15N2-labeled air to trace BNF activities in Δ anfH, Δ nifH, and Δ anfHΔ nifH mutants. Assessing bacterial growth, nitrogen content, and 15N incorporation revealed that BNF is impaired in K. radicincitans DSM16656T Δ nifH and Δ anfHΔ nifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants. [ABSTRACT FROM AUTHOR]

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A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains. [ABSTRACT FROM AUTHOR]

Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF–ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron-molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15 N 2 -labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15 N incorporation revealed that BNF is impaired in K. radicincitans DSM16656 T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656 T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.