Agmatine deiminase pathway genes in Lactobacillus brevis are linked to the tyrosine decarboxylation operon in a putative acid resistance locus In lactic acid bacteria (LAB), amino acids and their derivatives may be converted into aminecontaining compounds designated biogenic amines, in pathways providing metabolic energy and/ or acid resistance to the bacteria. In a previous study, a pathway converting tyrosine to tyramine was detected in Lactobacillus brevis and a fragment of a gene possibly involved in the production of another biogenic amine, putrescine, from agmatine, was detected in the same locus. The present study was carried out to determine if Lb. brevis actually harbours two biogenic amineproducing pathways in the same locus and to investigate the occurrence of the two gene clusters in other bacteria. Sequencing of the DNA locus in Lb. brevis revealed a cluster of six genes that are related to previously reported genes of agmatine deiminase pathways but with marked differences such as two genes encoding putative agmatine deiminases rather than one. Heterologous expression of encoded enzymes confirmed the presence of at least one active agmatine deiminase and one amino acid transporter that efficiently exchanged agmatine and putrescine. It was concluded that the Lb. brevis gene cluster encodes a functional and highly specific agmatine deiminase pathway. Screening of a collection of 197 LAB disclosed the same genes in 36 strains from six different species, and almost all the positive bacteria also contained the tyrosine catabolic pathway genes in the same locus. These results support the hypothesis that the agmatine deiminase and tyrosine catabolic pathways belong to a genomic region that provides acid resistance and that is exchanged horizontally as a whole between LAB. INTRODUCTIONLactic acid bacteria (LAB) can produce metabolic energy or increase their acid resistance by using catabolic pathways that convert amino acids or their derivatives into aminecontaining compounds designated biogenic amines (Molenaar et al., 1993; Fernández & Zú ñiga, 2006). In bacteria, biogenic amines may serve physiological functions such as cell proliferation control, but they are more generally released into the medium as the end products of the catabolic pathways (Tabor & Tabor, 1985;Griswold et al., 2006). In fact, excretion of the amine is an essential step in the pathways (see below). LAB carrying amino acid catabolic pathways are an important source of biogenic amines in foods (Ten Brink et al., 1990;Stratton et al., 1991;Silla Santos, 1996). There is interest in avoiding the proliferation of these bacteria because ingestion of foods containing large quantities of biogenic amines may cause human health issues such as headache, palpitations, flushing or vomiting (Silla Santos, 1996). Recently the genes of diverse pathways producing biogenic amines were identified in LAB (for a review, see Fernández & Zú ñiga, 2006). Interestingly, the pathways are strain specific rather than species specific, suggesting that horizonta...

The genome of the gram-positive bacterium Enterococcus faecalis contains the genes that encode the citrate lyase complex. This complex splits citrate into oxaloacetate and acetate and is involved in all the known anaerobic bacterial citrate fermentation pathways. Although citrate fermentation in E. faecalis has been investigated before, the regulation and transcriptional pattern of the cit locus has still not been fully explored. To fill this gap, in this paper we demonstrate that the GntR transcriptional regulator CitO is a novel positive regulator involved in the expression of the cit operons. The transcriptional analysis of the cit clusters revealed two divergent operons: citHO, which codes for the transporter (citH) and the regulatory protein (citO), and upstream from it and in the opposite direction the oadHDB-citCDEFX-oadA-citMG operon, which includes the citrate lyase subunits (citD, citE, and citF), the soluble oxaloacetate decarboxylase (citM), and also the genes encoding a putative oxaloacetate decarboxylase complex (oadB, oadA, oadD and oadH). This analysis also showed that both operons are specifically activated by the addition of citrate to the medium. In order to study the functional role of CitO, a mutant strain with an interrupted citO gene was constructed, causing a total loss of the ability to degrade citrate. Reintroduction of a functional copy of citO to the citO-deficient strain restored the response to citrate and the Cit ؉ phenotype. Furthermore, we present evidence that CitO binds to the cis-acting sequences O 1 and O 2 , located in the cit intergenic region, increasing its affinity for these binding sites when citrate is present and allowing the induction of both cit promoters.Enterococcus faecalis, a gram-positive catalase-negative coccus, is a natural member of the human and animal microflora. This ubiquitous microorganism has numerous fields of interest due to its importance as a cause of nosocomial infections and to its utilization in the food industry. With respect to the latter, the bacteria are involved in the ripening process and in aroma development of diverse cheeses. These positive effects have been attributed to specific biochemical traits such as lipolytic activity and citrate utilization. Citrate metabolism has been extensively studied in bacteria, both from a basic and applied point of view, since citrate fermentation plays an important role during the production of diverse kinds of drinks and foods (1-3, 7, 8, 12, 15, 16, 21, 28, 29, 31-33, 48-51).Citrate fermentation was recently investigated in E. faecalis, and the contribution to aroma development was characterized (18,46,47). In this microorganism, the first steps of the citrate degradative pathway are shared with other citrate-fermenting bacteria. Initially, citrate is converted to oxaloacetate and acetate by the enzyme citrate lyase. Then, oxaloacetate is decarboxylated to pyruvate. This compound is subsequently degraded to acetate, CO 2 , and formate with the generation of ATP by the acetate kinase enzyme. In addit...

Although the agmatine deiminase system (AgDI) has been investigated in Enterococcus faecalis, little information is available with respect to its gene regulation. In this study we demonstrate that the presence of exogenous agmatine induces the expression of agu genes in this bacterium. In contrast to the homologous and extensively characterized AgDI system of S. mutants, the aguBDAC operon in E. faecalis is not induced in response to low pH. In spite of this, agmatine catabolism in this bacterium contributes by neutralizing the external medium while enhancing bacterial growth. Our results indicate that carbon catabolic repression (CCR) operates on the AgDI system via a mechanism that involves interaction of CcpA and P-Ser-HPr with a cre site found in an unusual position considering the aguB promoter (55 nt upstream the +1 position). In addition, we found that components of the mannose phosphotransferase (PTSMan) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTSMan and CcpA double defective strain. Our gene context analysis revealed that aguR is present in oral and gastrointestinal microorganisms. Thus, regulation of the aguBDAC operon in E. faecalis seems to have evolved to obtain energy and resist low pH conditions in order to persist and colonize gastrointestinal niches.