In Klebsiella pneumoniae, six genes, constituting the pqqABCDEF operon, which are required for the synthesis of the cofactor pyrroloquinoline quinone (PQQ) have been identified. The role of each of these K. pneumoniae Pqq proteins was examined by expression of the cloned pqq genes in Escherichia coli, which cannot synthesize PQQ. All six pqq genes were required for PQQ biosynthesis and excretion into the medium in sufficient amounts to allow growth of E. coli on glucose via the PQQ-dependent glucose dehydrogenase. Mutants lacking the PqqB or PqqF protein synthesized small amounts of PQQ, however. PQQ synthesis was also studied in cell extracts. Extracts made from cells containing all Pqq proteins contained PQQ. Lack of each of the Pqq proteins except PqqB resulted in the absence of PQQ. Extracts lacking PqqB synthesized PQQ slowly. Complementation studies with extracts containing different Pqq proteins showed that an extract lacking PqqC synthesized an intermediate which was also detected in the culture medium of pqqC mutants. It is proposed that PqqC catalyzes the last step in PQQ biosynthesis. Studies with cells lacking PqqB suggest that the same intermediate might be accumulated in these mutants. By using pqq-lacZ protein fusions, it was shown that the expression of the putative precursor of PQQ, the small PqqA polypeptide, was much higher than that of the other Pqq proteins. Synthesis of PQQ most likely requires molecular oxygen, since PQQ was not synthesized under anaerobic conditions, although the pqq genes were expressed.

The plasmid-encoded citrate determinant of the Lactococcus lactis subsp. lactis var. diacetylactis NCDO176 was cloned and functionally expressed in a Cit-Escherichia coli K-12 strain. From deletion derivative analysis, a 3.4-kilobase region was identified which encodes the ability to transport citrate. Analysis of proteins encoded by the cloned fragment in a T7 expression system revealed a 32,000-dalton protein band, which correlated with the ability of cells to transport citrate. Energy-dependent [1,5-'4Cjcitrate transport was found with membrane vesicles prepared from E. coli cells harboring the citrate permease-expressing plasmid. The gene encoding citrate transport activity, citP, was located on the cloned fragment by introducing a site-specific mutation that abolished citrate transport and resulted in a truncated form of the 32,000-dalton expression product. The nucleotide sequence for a 2.2-kilobase fragment that includes the citP gene contained an open reading frame of 1,325 base pairs coding for a very hydrophobic protein of 442 amino acids, which shows no sequence homology with known citrate carriers.As in members of the family Enterobacteriaceae (25), the ability to utilize citrate is a useful metabolic characteristic for identifying Lactococcus lactis species (6,34). The citrate-fermenting ability of these gram-negative bacteria appears to be linked to the presence of genetically unstable determinants such as plasmids (13,14,18,32,33,37,38) (12,36,41), the energetics of citrate uptake are not yet understood. Kempler and McKay (19) demonstrated that the ability to transport citrate was linked to a 7.9-kilobase (kb) plasmid that appears to be present in all citrate-fermenting L. lactis strains analyzed. A detailed physical map of one of these citrate plasmids, pCT176, has been reported (10).In the bacterial species described until now, the ability to grow on citrate is associated with cation-dependent transport systems. Na+-dependent citrate utilization is found in Enterobacter aerogenes (16,28) and Salmonella typhimurium, which also possess a K+-dependent transport system (1,18,40 Media and growth conditions. E. coli strains were grown in L-broth (24) with vigorous shaking at 37°C. When appropriate, the medium was supplemented with carbenicillin (100 ,ug/ml), kanamycin (20 p,g/ml), or tetracycline (12.5 ,ug/ml) or a combination of these antibiotics.Citrate-positive recombinants of E. coli DH1 were selected after overnight incubation on Simmons citrate agar plates (Difco Laboratories).Cloning of the citP gene. CsCl-ethidium bromide density gradient-purified plasmid DNA from L. lactis NCDO176 was prepared by the method of Maniatis et al. (24) with minor variations as described previously (6) and was digested to completion with EcoRI. The 7.9-kb linearized plasmid band of pCT176 was isolated, inserted into the unique EcoRI site of vector pBR328, and transformed to E. coli MC1061.

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A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional j-galactosidase. Deletion and complementation analysis showed that the coding region for ,-galactosidase was located on a 5. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional 1-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N-and C-terminal parts, respectively, of ,-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral 13-galactosidase gene.Two systems for lactose transport and hydrolysis among bacteria are known. The first system has only been found in gram-positive bacteria and involves a phosphoenolpyruvatedependent phosphotransferase system, by which lactose is phosphorylated during transport and subsequently hydrolyzed by a phospho-,B-galactosidase (for reviews, see references 14 and 22). In the second, more widespread system, lactose is transported across the cellular membrane by a galactoside permease, and the unmodified internalized sugar is hydrolyzed by a 13-galactosidase. Most research has focused on the lactose permease (lacY) and ,-galactosidase (lacZ) genes from Escherichia coli (for reviews, see references 3, 24, and 25), and its lacZ gene has been developed into a useful tool in molecular genetics. Similar lac genes located on chromosomal or plasmid DNA have been found in other gram-negative bacteria (20), and in one instance a lac transposon (Tn951) has been reported (11).Recently, lac genes have been characterized in lactic acid bacteria that are used as starter cultures in dairy fermentations and therefore are highly specialized lactose utilizers. Genetic studies have shown that the lactose-specific phosphotransferase system enzymes are homologous and plasmid encoded in Lactococcus lactis (14)(15)(16)30) and Lactobacillus casei (1, 2, 38). In contrast, the homologous lac genes of Streptococcus thernophilus and Lactobacillus bulgaricus are chromosomally located and have been found to encode unique lactose permeases (28, 37) and ,B-galactosidases that show high similarity to those of gram-negative bacteria (42,43). A plasmid-encoded P-galactosidase in Lactobacillus casei ATCC 393 has been reported (10). In addition, we showed recently that Leuconostoc lactis NZ6009 also contains a lactose plasmid that codes for a P-galactosidase (13). gram-positive cocci that are used for industrial milk and wine fermentations. We recently started the genetic characterization of Leuconostoc spp. (13) and focused on the plasmidlocated lac genes in L. lactis NZ6009 (12). Here we describe the molecular characterization of a DNA fragment from the lactose plasmid pNZ63 that ...

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ObjectivesThe clinical and public health relevance of widespread case finding by testing for asymptomatic chlamydia infections is under debate. We wanted to explore future directions for chlamydia control and generate insights that might guide for evidence-based strategies. In particular, we wanted to know the extent to which we should pursue testing for asymptomatic infections at both genital and extragenital sites.MethodsWe synthesised findings from published literature and from discussions among national and international chlamydia experts during an invitational workshop. We described changing perceptions in chlamydia control to inform the development of recommendations for future avenues for chlamydia control in the Netherlands.ResultsDespite implementing a range of interventions to control chlamydia, there is no practice-based evidence that population prevalence can be reduced by screening programmes or widespread opportunistic testing. There is limited evidence about the beneficial effect of testing on pelvic inflammatory disease prevention. The risk of tubal factor infertility resulting from chlamydia infection is low and evidence on the preventable fraction remains uncertain. Overdiagnosis and overtreatment with antibiotics for self-limiting and non-viable infections have contributed to antimicrobial resistance in other pathogens and may affect oral, anal and genital microbiota. These changing insights could affect the outcome of previous cost–effectiveness analysis.ConclusionThe balance between benefits and harms of widespread testing to detect asymptomatic chlamydia infections is changing. The opinion of our expert group deviates from the existing paradigm of ‘test and treat’ and suggests that future strategies should reduce, rather than expand, the role of widespread testing for asymptomatic chlamydia infections.

The citrate permease determinant (citP) in several Leuconostoc strains was demonstrated to be plasmid encoded by curing experiments and hybridization studies with a DNA fragment containing the citP gene from Lactococcus lactis subsp. lactis biovar diacetylactis NCDO176. Cloning and nucleotide sequence analysis of Leuconostoc lactis NZ6070 citP revealed almost complete identity to lactococcal citP.

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