Abstract: Method for producing multiple modifications in the chromosome of Gram-negative bacteria and Salmonella strains which are deficient in c-di-GMP synthesis obtained by said method. The method can be used to make multiple modifications in the genome of Gram-negative bacteria, simply and efficiently, using the plasmids of the invention, which comprise a marker gene under the control of a constitutive promoter, a replication origin specific for Gram-negative bacteria, a gene which encodes a heat-sensitive protein essential for initiating replication of the plasmid, making the replication origin heat-sensitive, and a counter-selection gene. The invention also relates to mutant Salmonella enterica strains obtained using the method of the invention in which some of the twelve genes which encode proteins with GGDEF domain have been selected*, and to the use thereof as expression vectors, immunotherapeutic agents and in metabolic studies.

Abstract: Method for producing multiple modifications in the chromosome of Gram-negative bacteria and Salmonella strains which are deficient in c-di-GMP synthesis obtained by said method. The method can be used to make multiple modifications in the genome of Gram-negative bacteria, simply and efficiently, using the plasmids of the invention, which comprise a marker gene under the control of a constitutive promoter, a replication origin specific for Gram-negative bacteria, a gene which encodes a heat-sensitive protein essential for initiating replication of the plasmid, making the replication origin heat-sensitive, and a counter-selection gene.