Complete DNA sequence of Lactococcus lactis adds flavor to genomics.

Abstract

With the publication of the longawaited genome sequence of Lactococcus lactis ssp. lactis IL1403, Bolotin et al. (2001) have set the stage for functional and comparative genomics of an important group of industrial microorganisms, that is, the lactic acid bacteria (LAB). LAB are widely used for starting industrial fermentations of milk, vegetables, meat, and fish. L. lactis is used in cheesemaking, as it is involved in casein degradation, in acidification by formation of lactate, and in the formation of flavor compounds. Other LAB, such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and certain Bifidobacteria, are used for yogurt production, whereas Proprionibacteria and other species contribute to the product characteristics of Swiss-type cheeses. L. lactis is the major starter for Cheddar production; for a Dutch cheese such as Gouda, a complex starter culture containing >100 different strains of LAB is used. L. lactis is by far the best characterized LAB with respect to its physiology, metabolic pathways, and regulatory mechanisms. The ease of genetic modification of various species of LAB and the existence of a worldwide network of LAB researchers have greatly stimulated fundamental and applied research in LAB (Gasson and de Vos 1994; Konings et al. 1999). Industrial applications are directed mainly toward improvement of flavor, texture, and preservation characteristics of fermented products as well as enhancement of industrial robustness of starter cultures. Novel uses of LAB, such as in oral vaccines or in preparation of functional foods or their direct application as a probiotic culture, are also being explored (Delcour et al. 1999). For oral vaccines, specific antigenic determinants are overproduced either intraor extracellularly in LAB and subsequently administered orally, nasally, or vaginally to the animal to stimulate mucosal immunity (Gilbert et al. 2000). In situ production of interleukin in mice has already been successful, showing the high potential of LAB for medical applications (Steidler et al. 2000). The main breakthroughs for using L. lactis as an improved cell factory were the development of a wide variety of genetic modification tools, highly effective controlled-gene expression systems, novel metabolic engineering strategies, and food-grade cloning systems (Kuipers et al. 2000). In this respect, all requirements are fulfilled to make optimal use of the information to be mined from the lactococcal genome. The investigators from INRA and Genoscope have used an original approach, comprising diagnostic sequencing followed by a shotgun polishing step. This has resulted in a 2.37-Mb genome sequence, which upon annotation revealed 2310 proteins. The error rate in the final sequence is stated to be <0.01%. The L. lactis genome was shown to contain quite high numbers of IS elements and prophages (Chopin et al. 2001), as expected from earlier work. It also contains the relatively high number of paralogous genes that are found in, for example, Bacillus subtilis, the first fully sequenced gram-positive bacterium (Kunst et al. 1997). However, only three putative -factors were found in L. lactis in contrast to the 18 identified in B. subtilis, which indicates that regulation of expression of many genes is achieved quite differently in both organisms. Moreover, only eight twocomponent regulatory systems (instead of 34 in B. subtilis) were found, indicating that L. lactis may be less wellequipped to adjust to changing environmental conditions, although several mechanisms for dealing with stress have been described (Rallu et al. 2000). Actually, L. lactis resides in a much more stable nutritional environment than does B. subtilis, obviating the need for an extensive adaptation machinery. A particularly interesting feature in the annotated sequence is the occurrence of genes usually related to competence, that is, the natural ability to take up DNA from the environment. Unlike the situation in B. subtilis, all latecompetence genes in L. lactis possess leaderless mRNAs, indicating a quite different type of regulation of their expression. It remains to be shown if all genes required for competence development are present and under which conditions L. lactis would be able to become competent. With the aid of the current knowledge of other competent bacteria such as B. subtilis and Streptococcus pneumoniae, one could expect these questions to be answered soon. The finding that several genes involved in aerobic respiration were present, including men, hemHKN, and cytABCD operons, is rather surprising. In conjunction with the notion that improved growth occurs in media containing hemin, this suggests that aerobic respiration does exist in this fermentative bacterium. Bolotin et al. (2001) describe many more interesting features related to protein secretion, cell wall metabolism, and putative horizontal gene transfer between Lactococci and certain gramnegative bacteria, which forms the start E-MAIL o.p.kuipers@biol.rug.nl; FAX 0031503632348. Article and publication are at www.genome.org/cgi/ doi/10.1101/gr.188501. Insight/Outlook