Annette Arndt

The Alcohol Dehydrogenase Gene adhA in Corynebacterium glutamicum Is Subject to Carbon Catabolite Repression

Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene (adhA), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum. Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA, and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose- and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA.

Ethanol Catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h–1 and biomass yields up to 0.47 g dry weight (g ethanol)–1. Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.

RamA and RamB are global transcriptional regulators in Corynebacterium glutamicum and control genes for enzymes of the central metabolism

In Corynebacterium glutamicum, the transcriptional regulators of acetate metabolism RamA (encoded by cg2831) and RamB (encoded by cg0444) play an important role in expression control of genes involved in acetate and ethanol metabolism. Both regulators were speculated to have broader significance in expression control of further genes in the central metabolism of C. glutamicum. Here we investigated the RamA and RamB regulons by genome-wide transcriptome analysis with special emphasis on genes encoding enzymes of the central carbon metabolism. When compared to the parental wild-type, 253 genes and 81 genes showed different mRNA levels in defined RamA- and RamB-deficient C. glutamicum strains, respectively. Among these were genes involved in sugar uptake, glycolysis, gluconeogenesis, acetate, l-lactate or ethanol metabolism. The direct interaction of RamA and RamB proteins with the respective promoter/operator fragments was demonstrated in vitro by electrophoretic mobility shift assays. Taken together, we present evidence for an important role of RamA and RamB in global gene expression control in C. glutamicum.

Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB

Corynebacterium glutamicum has been shown to grow with ethanol as the sole or as additional carbon and energy source and accordingly, to possess both alcohol dehydrogenase and acetaldehyde dehydrogenase (ALDH) activities, which are responsible for the two-step ethanol oxidation to acetate. Here we identify and functionally analyze the C. glutamicum ALDH gene (cg3096, ald), its expression and its regulation. Directed inactivation of the chromosomal ald gene led to the absence of detectable ALDH activity and to the inability to grow on or to utilize ethanol, indicating that the ald gene product is essential for ethanol metabolism and that no ALDH isoenzymes are present in C. glutamicum. Transcriptional analysis revealed that ald from C. glutamicum is monocistronic, that ald transcription is initiated 92 nucleotides upstream of the translational start codon ATG and that ald expression is much lower in the presence of glucose in the growth medium. Further analysis revealed that transcription of the ald gene is under control of the transcriptional regulators RamA and RamB. Both these proteins directly bind to the respective promoter region, RamA is essential for expression and RamB exerts a slightly negative effect on ald expression on all carbon sources tested.

l-Valine Production during Growth of Pyruvate Dehydrogenase Complex- Deficient Corynebacterium glutamicum in the Presence of Ethanol or by Inactivation of the Transcriptional Regulator SugR

ABSTRACT Pyruvate dehydrogenase complex-deficient strains of Corynebacterium glutamicum produce l-valine from glucose only after depletion of the acetate required for growth. Here we show that inactivation of the DeoR-type transcriptional regulator SugR or replacement of acetate by ethanol already in course of the growth phase results in efficient l-valine production.

A streptococcal effector protein that inhibits Porphyromonas gingivalis biofilm development.

Dental plaque formation is a developmental process involving cooperation and competition within a diverse microbial community, approximately 70 % of which is composed of an array of streptococci during the early stages of supragingival plaque formation. In this study, 79 cell-free culture supernatants from a variety of oral streptococci were screened to identify extracellular compounds that inhibit biofilm formation by the oral anaerobe Porphyromonas gingivalis strain 381. The majority of the streptococcal supernatants (61 isolates) resulted in lysis of P. gingivalis cells, and some (17 isolates) had no effect on cell viability, growth or biofilm formation. One strain, however, produced a supernatant that abolished biofilm formation without affecting growth rate. Analysis of this activity led to the discovery that a 48 kDa protein was responsible for the inhibition. Protein sequence identification and enzyme activity assays identified the effector protein as an arginine deiminase. To identify the mechanism(s) by which this protein inhibits biofilm formation, we began by examining the expression levels of genes encoding fimbrial subunits; surface structures known to be involved in biofilm development. Quantitative RT-PCR analysis revealed that exposure of P. gingivalis cells to this protein for 1 h resulted in the downregulation of genes encoding proteins that are the major subunits of two distinct types of thin, single-stranded fimbriae (fimA and mfa1). Furthermore, this downregulation occurred in the absence of arginine deiminase enzymic activity. Hence, our data indicate that P. gingivalis can sense this extracellular protein, produced by an oral streptococcus (Streptococcus intermedius), and respond by downregulating expression of cell-surface appendages required for attachment and biofilm development.

HU protein affects transcription of surface polysaccharide synthesis genes in Porphyromonas gingivalis.

K-antigen capsule synthesis is an important virulence determinant of the oral anaerobe Porphyromonas gingivalis. We previously reported that the locus required for synthesis of this surface polysaccharide in strain W83 (TIGR identification PG0106 to PG0120) is transcribed as a large (∼16.7-kb) polycistronic message. Through sequence analysis, we have now identified a 77-bp inverted repeat located upstream (206 bp) of the start codon of PG0106 that is capable of forming a large hairpin structure. Further sequence analysis just upstream and downstream of the capsule synthesis genes revealed the presence of two genes oriented in the same direction as the operon that are predicted to encode DNA binding proteins: PG0104, which is highly similar (57%) to DNA topoisomerase III, and PG0121, which has high similarity (72%) to DNA binding protein HU (β-subunit). In this report, we show that these two genes, as well as the 77-bp inverted repeat region, are cotranscribed with the capsule synthesis genes, resulting in a large transcript that is ∼19.4 kb (based on annotation). We also show that a PG0121 recombinant protein is a nonspecific DNA binding protein with strong affinity to the hairpin structure, in vitro, and that transcript levels of the capsule synthesis genes are downregulated in a PG0121 deletion mutant. Furthermore, we show that this decrease in transcript levels corresponds to a decrease in the amount of polysaccharide produced. Interestingly, expression analysis of another polysaccharide synthesis locus (PG1136 to PG1143) encoding genes involved in synthesis of a surface-associated phosphorylated branched mannan (APS) indicated that this locus is also downregulated in the PG0121 mutant. Altogether our data indicate that HU protein modulates expression of surface polysaccharides in P. gingivalis strain W83.

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoid-associated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUα (PG1258) and the HUβ (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUβ using comparative expression profiling of the PG0121 (HUβ) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUβ has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake.

Control of adhA and sucR expression by the SucR regulator in Corynebacterium glutamicum.

The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to a complex carbon source-dependent regulation mediated by RamA, RamB and GlxR. In this study we identified SucR as the fourth transcriptional regulator involved in expression control of the adhA gene. SucR specifically binds to the adhA promoter and acts as transcriptional repressor independent of the carbon source used. Furthermore, we found that SucR negatively controls the expression of its own gene. This negative autoregulation is mediated by binding of SucR to at least one of four identified binding sites located in the promoter region of sucR. EMSA experiments and subsequent sequence analysis led to the identification of the SucR consensus binding sequence YYAACAWMAW. This binding motif is different from the binding site (ACTCTAGGGG) recently described for SucR in the promoter region of the sucCD operon. However, we were not able to detect a specific interaction of purified SucR protein with this motif present in the sucCD promoter region.