Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Chromosomal mutations causing resistance to tetracycline in Bacillus subtilis.

Summary We have isolated, after ethylmethanesulfonate mutagenesis, several chromosomal mutations causing resistance to tetracycline in Bacillus subtilis. These mutations fall into two classes, tetA and tetB. 30 S ribosomal protein S10 shows an altered mobility on two-dimensional acrylamide gels in cells bearing the former type of mutation. Ribosomes from these cells show elevated levels of resistance to tetracycline in vitro as measured by polyuridine dependent polyphenylalanine synthesis. The tetA locus maps adjacent to the tuf gene in the B. subtilis ribosomal protein gene cluster. Cells with the tetB mutation do not show any altered ribosomal protein, and their ribosomes are as sensitive, in vitro, to tetracycline as ribosomes isolated from wild type cells. The tetB mutation has been mapped proximal to cysA14.In partial fulfillment of the requirements for the doctoral degree by G.W. in the Department of Biology at the New York University Graduate School of Arts and Sciences     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Complex regulation of the Bacillus subtilis aconitase gene

The roles of the CcpC, CodY, and AbrB proteins in regulation of the Bacillus subtilis aconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulating citB expression in minimal-glucose-glutamine medium, also contributed to repression of citB during exponential-phase growth in broth medium. A null mutation in codY had no effect on citB expression during growth in minimal medium even when combined with ccpC and abrB mutations. However, a codY mutation slightly relieved repression during exponential growth in broth medium and completely derepressed citB expression when combined with a ccpC mutation. An abrB mutation led to decreased expression of citB during stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within the citB regulatory region. Interaction of CcpC and CodY with the citB promoter region was partially competitive.     

Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system

The tetracycline repressor (TetR) regulates the most abundant resistance mechanism against the antibiotic tetracycline in grain-negative bacteria. The TetR protein and its mutants are commonly used as control elements to regulate gene expression in higher eukaryotes. We present the crystal structure of the TetR homodimer in complex with its palindromic DNA operator at 2.5 A resolution. Comparison to the structure of TetR in complex with the inducer tetracycline-Mg2+ allows the mechanism of induction to be deduced. Inducer binding in the repressor core initiates conformational changes starting with C-terminal unwinding and shifting of the short helix a6 in each monomer. This forces a pendulum-like motion of helix a4, which increases the separation of the attached DNA binding domains by 3 A, abolishing the affinity of TetR for its operator DNA.     

Osmotic pressure can regulate matrix gene expression in Bacillus subtilis

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non‐specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non‐specifically co‐ordinate the behaviour of cells in communities composed of many different species.