The genetic manageability of the biotechnologically important is hampered due to

The genetic manageability of the biotechnologically important is hampered due to its poor transformability whereas efficiently takes up DNA during genetic competence a quorum-sensing-dependent process. ComK is VEGFA usually inhibited at low cell densities by PHA-680632 a proteolytic complex in which MecA binds ComK and such inhibition is usually antagonized by the conversation of MecA with ComS (the expression of the latter is controlled by cell density in analysis of MecA and the hitherto unidentified ComS which revealed differences for qualified and noncompetent strains indicating that the reduced competence possibly is due to a nonfunctional coupling of the (a generally-regarded-as-safe [GRAS] species) serve as microbial workhorses as they produce a quantity of useful compounds (10) and they possess a high capacity for the secretion of exoenzymes such as amylases and proteases with yields of up to 25 g liter?1 PHA-680632 (46). The availability of the genome sequences of strain DSM13 (61) and the isogenic ATCC 14580 (44) already facilitated a number of developments aiming at strain improvement and the enhancement of biosafety (38 63 64 However the low frequency of transformation regularly observed with those strains is usually a drawback. Although a derivative (MW3) was obtained by the deletion of the genes encoding type I restriction enzymes allowing the routine creation of transformants PHA-680632 by protoplast transformation it still has a rather low transformation efficiency (65). Hence the generation of mutants by homologous recombination is usually a rather time-consuming and cumbersome process as is the case for other members of the genus (60). During the a part of their life cycle when they develop natural genetic competence representatives of the genus generally are capable of taking up exogenously supplied DNA. Such a DNA uptake mechanism was first explained for (49) and frequently has been used to obtain transformants and generate deletions and conditional mutants with inducible gene expression (58). The establishment of such a system in DSM13 would considerably improve PHA-680632 genetic handling in this industrial workhorse. The key regulator responsible for the development of genetic competence is usually ComK controlling the transcription of all genes involved in DNA binding processing uptake and homologous recombination between the incoming and the host DNA (16 20 ComK expression is turned down during exponential growth and a number of stimuli must be integrated to control expression (Fig. ?(Fig.1)1) (14 18 23 40 In 168 (27) and FZB42 (7) the quorum-sensing-dependent escape from ComK proteolysis depends on a regulatory operon containing four genes (20). ComQ is responsible for processing pre-ComX to generate the active ComX peptide pheromone (1). The accumulation of extracellular ComX is usually sensed by sensor histidine kinase ComP which is usually capable of phosphorylating the cognate response regulator ComA. Phosphorylated ComA (ComA~P) induces the transcription of the operon which codes for the biosynthetic pathway of the biotenside surfactin and which includes the 47 amino acids spanning ComS peptide (34). ComS prevents the proteolysis of ComK by competitively binding to MecA thereby blocking the proteolytic MecA/ClpCP complex (13 17 31 43 57 Furthermore ComA~P also facilitates the transcription of by promoting the phosphorylation of DegQ and increases the amount of DegU~P which facilitates the synthesis of extracellular enzymes and polyglutamate production (Fig. ?(Fig.1)1) (8 35 39 50 FIG. 1. Regulation of expression (only proteins and loci resolved in this work are pointed out). ComK activates the transcription of its own gene forming a positive autoregulatory loop (30 47 The DegS/DegU two-component system influences competence development … However ATCC 14580 and the isogenic DSM13 carry an insertion element within the locus. Hence poor genetic competence in DSM13 may be due to the lack of a functional ComP. Since natural genetic competence has been reported to occur in 9945A (32 54 it was considered that understanding the lack of natural genetic competence in strain DSM13 and derivatives could lead to the development of qualified strains and thus make efficient genetic tools available. MATERIALS AND METHODS Bioinformatic sequence analysis. Sequence alignments were done with CLUSTALW (53). Visual alignments of GC contents were performed with Artemis (5). Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are outlined in Table ?Table11 ..