SummaryAmtR, the master regulator of nitrogen control in Corynebacterium glutamicum , represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pulldown assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR.The AmtR regulon was characterized in this study by a combination of bioinformatics, transcriptome and proteome analyses. At least 33 genes are directly controlled by the repressor protein including those encoding transporters and enzymes for ammonium assimilation ( amtA , amtB , glnA , gltBD ), urea and creatinine metabolism ( urtABCDE , ureABCEFGD , crnT , codA ), a number of biochemically uncharacterized enzymes and transport systems (NCgl1099, NCgl1100, NCgl 1915-1918) as well as signal transduction proteins ( glnD , glnK ). For the AmtR regulon, an AmtR box has been defined which comprises the sequence tttCTATN 6 AtAGat/aA. Furthermore, the transcriptional organization of AmtR-regulated genes and operons was characterized.

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.

The Corynebacterium glutamicum gltB and gltD genes, encoding the large (α) and small (β) subunit of glutamate synthase (GOGAT), were investigated in this study. Using RT-PCR, a common transcript of gltB and gltD was shown. Reporter gene assays and Northern hybridization experiments revealed that transcription of this operon depends on nitrogen starvation. The expression of gltBD is under control of the global repressor protein AmtR as demonstrated by gel shift experiments and analysis of gltB transcription in an amtR deletion strain. In contrast to other bacteria, in C. glutamicum GOGAT plays no pivotal role ; e.g. gltB and gltD inactivation did not result in growth defects when cells were grown in standard minimal medium and only a slight increase in the doubling time of the corresponding mutant strains was observed in the presence of limiting amounts of ammonia or urea. Additionally, mutant analyses revealed that GOGAT has no essential function in glutamate production by C. glutamicum.

The molecular identification of the Corynebacterium glutamicum urea uptake system is described. This ABC-type transporter is encoded by the urtABCDE operon, which is transcribed in response to nitrogen limitation. Expression of the urt genes is regulated by the global nitrogen regulator AmtR, and an amtR deletion strain showed constitutive expression of the urtABCDE genes. The AmtR repressor protein also controls transcription of the urease-encoding ureABCEFGD genes in C. glutamicum. The ure gene cluster forms an operon which is mainly transcribed in response to nitrogen starvation. To confirm the increased synthesis of urease subunits under nitrogen limitation, proteome analyses of cytoplasmic protein extracts from cells grown under nitrogen surplus and nitrogen limitation were carried out, and five of the seven urease subunits were identified.Urea is a readily available nitrogen source, since it is excreted by a variety of organisms into the environment. In bacteria able to metabolize this solute, urea is hydrolyzed by the cytoplasmic urease enzyme complex, leading to one CO 2 and two ammonium molecules (for review, see reference 20). Although urea is small and uncharged and can therefore easily pass the bacterial membrane, many prokaryotes, including Corynebacterium glutamicum, synthesize energy-dependent transport systems for its uptake. When present in high concentrations, sufficient urea crosses the C. glutamicum cytoplasmic membrane by passive diffusion, and only under conditions of nitrogen starvation is an energy-dependent urea uptake system synthesized (27). With a K m of 8 M for urea, the affinity of this uptake system is much higher than the affinity of urease for its substrate (K m of approximately 55 mM). The maximum urea uptake rate depends on the level of expression and is relatively low at 2.0 to 3.5 nmol mg (dry weight) Ϫ1 min Ϫ1 (27). Genes coding for the C. glutamicum urea uptake system have not been previously identified.The genes encoding the urease enzyme complex were isolated and sequenced (21, 24). While ureA, ureB, and ureC encode the urease structural subunits, the ureE, ureF, ureG, and ureD genes code for accessory proteins. As shown by mutant analyses, at least the ureC and the ureD products are essential for a functional urease (21). Depending on the growth conditions, urease activity observed in the wild type varies between 0.9 and 2.2 U mg of protein Ϫ1 for cells grown in ammonium-rich minimal medium (21, 24), between 1.0 and 1.6 U mg of protein Ϫ1 when glutamine is used as nitrogen source (24), and between 2.0 and 6.1 U mg of protein Ϫ1 when different concentrations of urea were added to the medium as sole nitrogen source (21, 24). These medium-dependent changes in urease activity indicate that the enzyme is moderately regulated, either on the level of activity or on the level of gene expression.Here, we describe the identification of genes encoding the C. glutamicum urea uptake system and their transcriptional organization and regulation, as well as the organization and control ...

BackgroundParents are the ones who decide whether or not to participate in parent focused prevention trials. Their decisions may be affected by internal factors (e.g., personality, attitudes, sociodemographic characteristics) or external barriers. Some of these barriers are study-related and others are intervention-related. Internal as well as external barriers are especially important at the screening stage, which aims to identify children and families at risk and for whom the indicated prevention programs are designed. Few studies have reported their screening procedure in detail or analyzed differences between participants and dropouts or predictors of dropout. Rates of participation in prevention programs are also of interest and are an important contributor to the efficacy of a prevention procedure.MethodsIn this study, we analyzed the process of parent recruitment within an efficacy study of the indicated Prevention Program for Externalizing Problem behavior (PEP). We determined the retention rate at each step of the study, and examined differences between participants and dropouts/decliners. Predictors of dropout at each step were identified using logistic regression.ResultsRetention rates at the different steps during the course of the trial from screening to participation in the training ranged from 63.8% (pre-test) to 81.1% (participation in more than 50% of the training sessions). Parents who dropped out of the study were characterized by having a child with lower symptom intensity by parent rating but higher ratings by teachers in most cases. Low socioeconomic status and related variables were also identified as predictors of dropout in the screening (first step) and for training intensity (last step).ConclusionsSpecial attention should be paid to families at increased risk for non-participation when implementing the prevention program in routine care settings.Trial RegistrationISRCTN12686222

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, the use of creatinine as an alternative nitrogen source in Corynebacterium glutamicum, the identification of a membrane protein involved in creatinine uptake, the transcriptional regulation of the corresponding gene, and expression regulation of the gene encoding the creatinine deaminase are reported. As shown by mutant analyses, RNA hybridization experiments and real-time PCR, the expression of two genes, crnT and codA, is increased in response to nitrogen limitation, and regulation depends on the global nitrogen regulator AmtR. In addition, synthesis of creatinine deaminase during nitrogen starvation was shown by two-dimensional gel electrophoresis and MALDI-TOF-MS followed by peptide mass fingerprint analysis.

The Corynebacterium glutamicum genes encoding urease were isolated and sequenced. While ureA, ureB and ureC are encoding structural subunits of urease, ureE, ureF, ureG and ureD are encoding accessory proteins. As deduced from DNA sequence analyses, the ure genes are transcriptionally coupled, this was proven by RT-PCR at least for ureABC. Gene disruption experiments revealed that both structural (UreC) and accessory proteins (UreD) are indispensable for urease activity and growth on urea. Urease activity was determined in different Corynebacterium species after growth in various media. While the regulation patterns observed revealed species-specific differences, in general urease activity is induced upon nitrogen starvation. As in mycobacteria, in corynebacteria urease activity was highest in a pathogenic species and might also play a role in host-pathogen interaction.

Genes encoding proteins for ammonium uptake, assimilation, and the nitrogen regulatory system in Corynebacterium diphtheriae were studied on basis of homology searches using Corynebacterium glutamicum genes as query sequences. Regulation of transcription of these genes in response to nitrogen starvation was analyzed by RNA hybridization experiments and knock-out mutants were generated to verify the function of distinct genes. In this communication, we were able to identify the key components of ammonium assimilation pathways and nitrogen regulation in C. diphtheriae. Moreover, we show in this study that molecular biology methods and vectors developed for C. glutamicum can be applied in C. diphtheriae. The results obtained strengthens the role of C. glutamicum as a model organism for mycolic acids-containing actinomycetes.