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Gene duplication and horizontal gene transfer play an important role in the evolution of prokaryotic genomes. We have investigated the role of three CprK paralogues from the cAMP receptor protein-fumarate and nitrate reduction regulator (CRP-FNR) family of transcriptional regulators that are encoded in the genome of Desulfitobacterium hafniense DCB-2 and possibly regulate expression of genes involved in the energy-conserving terminal reduction of organohalides (halorespiration). The results from in vivo and in vitro promoter probe assays show that two regulators (CprK1 and CprK2) have an at least partially overlapping effector specificity, with preference for ortho-chlorophenols, while meta-chlorophenols proved to be effectors for CprK4. The presence of a potential transposase-encoding gene in the vicinity of the cprK genes indicates that their redundancy is probably caused by mobile genetic elements. The CprK paralogues activated transcription from promoters containing a 14 bp inverted repeat (dehalobox) that closely resembles the FNR-box. We found a strong negative correlation between the rate of transcriptional activation and the number of nucleotide changes from the optimal dehalobox sequence (TTAAT-[N.sub.4]-ATTAA). Transcription was initiated by CprK4 from a promoter that is situated upstream of a gene encoding a methyl-accepting chemotaxis protein. This might be the first indication of taxis of an anaerobic bacterium to halogenated aromatic compounds.

A chemically defined medium in combination with an airlift fermentor system is used to study the growth and sporulation of Bacillus cereus ATCC 14579. Findings reveal that Bacillus cereus is able to form spores on a chemically defined medium without glucose but with lactate as a main carbon source.

The nucleotide sequence analyses and characterization of the three plasmids from Lactobacillus plantarum WCFS1 are described. Cloning vectors were constructed based on the replicas of all three plasmids and out of this plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8.

The conditions for growth and sporulation of Bacillus cereus ATCC 14579, which has been characterized at genome level, in a chemically defined medium with an airlift fermentor system are described. The growth and sporulation can be monitored and manipulated so that a synchronous growth and sporulation occurs.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835) , analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N -acetylglucosamine. Some of the N -acetylmuramic acid (MurNAc) subunits were O -acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non- N -acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host–microbiome interactions. [ABSTRACT FROM AUTHOR]

Research has been conducted on lactic acid bacteria which contain alr gene encoding alanine racemase. The use of this gene as a heterologous food-grade selection marker in lactic acid bacteria is described.

Research has been conducted on Bifidobacterium lactis and B. adolescentis. The viabily of these bacteria during bile salt stress exposure has been tested, and the use of carboxyfluorescein diacetate, oxonol and propidium iodide in monitoring esterase activity and membrane integrity as bacterial viability indicators is described.

The authors review the presentations carried out by the researches at the Sixth International Conference on Streptococcal, Lactococcal and Enterococcal Genetics convened in 2002 in Asheville, N.C. The topics of interest include the role of streptococcal, lactococcal and enterococcal species in bacterial genetics development and the discovery and analysis of yet-to-be-cultured microbes and microbes colonizing oral cavity, respiratory tract and intestinal system.

Research has been conducted on the uncultured Ruminococcus obeum-like bacteria in human feces. The probes developed to detect and quantify these bacteria via fluorescent in situ hybridization, flow cytometry and direct microscopy are described.

Research has been conducted on the hyperthermophilic archaeon Pyrococcus sp. strain JT1. The identification of the plasmid in this archaeon has been carried out and the presence of the DNA replication intermediates of the plasmid have been detected.

Research has been conducted on the microbial communities in the artisanal fermented food product, Ragusano cheese. The cultivation-independent molecular methods developed in analyzing the microbial population dynamics and diversity during cheese manufacturing are described.

Research has been conducted on the beta-mannosidase gene from the Pyrococcus horikoshii genome. The cloning and expression of this gene in Escherichia coli have been carried out and the characterization results of the gene product beta-mannosidase are presented.

The polymerase chain reaction and denaturing gradient gel electrophoresis of 16S ribosomal DNA can be used to identify bacteria in the human gastrointestinal tract. Using this method, researchers identified four Leuconostoc species that have never before been found in the GI tract.

Phylogenetic analysis suggests that the ADP-dependent phosphofructokinase is a key enzyme of the modified Emden-Meyerhof pathway in mesophilic and thermophilic methanogenic archaea. Kinetic analysis show low enzyme activity with ADP and acetyl-phosphate serving as phosphoryl donors.

The 7-kDa cold shock proteins of Lactococcus lactis MG1363 not only protect the bacterium from low temperatures but also regulate the production of non-7-kDa cold-induced proteins. Lactic acid bacteria are used in industrial fermentation and they undergo a variety of temperature changes during this process.

Researchers have genetically engineered Clostridium beijeinckii with glycoside hydrolase genes from the rumen fungus Neocallimastix patriciarum that allow C. beijerinckii to grow on lichenan. Clostridium spp. can produce butanol and ethanol from carbohydrates and biomass is the most widely available substrate for this conversion.

Research has been conducted on the Desulfitobacterium dehalogenans's batch cultures grown on different electron donors. The amount of energy conserved via the halorespiration by these cultures has been determined, and the results of the growth yields obtained from the batch cultures have been compared.

Overexpression of the UPD-glucose pyrophosphorylase (galU) gene of Lactococcus lactis MG1363 under control of the nisA promoter causes an increase in GalU activity and increases in UDP-glucose and UDP-galactose. UDP-glucose and UDP-galactose are precursors of exopolysaccharides.

An electroporation-based transformation procedure to create an efficient host-vector system in Desulfitobacterium dehalogenans is described. This microorganism can degrade pollutants in soils, aquifers, and sediments.

Researchers amplified a 520-bp fragment from 16S ribosomal DNA of Bifidoacterium and then separated the fragments using denaturing gradient gel electrophoresis. This technique was used to identify Bifidobacterium species in human fecal samples.

Research on Lactococus lactis has produced clues for methods to increase exopolysaccharide (EPS) production via genetic manipulation. Nuclear magnetic resonance was used to determine phosphorylated intermediate concentrations in cell wall and EPS biosynthetic pathways; metabolic targets include an increase in pool size of the lipid carrier and an enhancement of EPS precursor production flux.

Results indicate that composition of bacterial ribosome types exhibit definite shifts in response to grassland succession. An initial increase in ribosome level following cessation of agricultural production and fertilization correlated with the collapse of Lolium perenne population and organic matter mineralization. Data further show positive correlation between soil bacterial activity and ribosome level.

Research has been conducted on the substrate specificity of the beta-glucosidase from Pyrococcus furiosus. The use of X-ray diffraction and crystallization is described.

Results demonstrate that archaea bacterial alanine aminotransferase is a dimer consisting of a two identical subunits of 46 kilodaltons. Data indicate that production of L-alanine from fermentative metabolism mediated by the enzyme is an ancestral metabolic characteristic in archaea bacteria.

Research has been conducted on the hyperthermophilic archaeon Pyrococcus furiosus beta-glucosidase. A library of random CelB mutants in Escherichia coli has been generated.

Research was conducted to analyze halorespiration at molecular level in gram-positive bacterial mutants derived from transposon Tn916 mutagenesis. Results indicate that the genes encoding different respiratory elements in the mutants are disrupted.

Streptococcus thermophilus CNRZ302 is discussed relative to its cold shock proteins and low-temperature response. Evidence for an active adaptation response of the thermophilic starter LAB S. thermophilus to a freezing challenge after exposure to low temperature has been found.

A study concerning the chemical composition of exopolysaccharide (EPS)- producing Lactococcus lactis strains is presented. Experiments include investigation of eps genes locations, sequences and organization.

The article describes acetate and glucose cometabolism in a L-lactate dehydrogenase-deficient strain of Lactococcus lactis where acetate is converted to ethanol. Alternative pathway for NAD (super)+ regeneration is described.

A study was conducted to examine the genetic organization, expression and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus. Results indicate that the celB locus is composed of the celB gene, which codes for an intracellular beta-glucosidase, and a divergently oriented gene cluster labeled adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase. It was also found that transcription of both the celB gene and the lamA operon is increased with the introduction of glucose-containing substrates.

A study was conducted to analyze disruptions in several genes of the nisin gene cluster of Lactococcus lactis and their impact on the production of nisin and producer immunity. The disruption of the NisB gene was carried out using an integrative plasmid. Results indicated that the Nisl gene supports a critical function in nisin immunity and cooperates with nisFEG-encoded proteins to promote a high level of immunity.

The molecular characterization of the Lactococcus lactis ptsHI operon and analysis of the regulatory role of HPr were conducted. The L. lactis ptsH and ptsI genes were cloned and a promoter sequence was identified upstream of the ptsHI operon. Results demonstrate the role of HPr(Ser-P) as corepressor in the catabolite repression of the gal operon. Findings also reveal that HPr(Ser-P) acts as a coactivator in the CcpA-mediated catabolite activation of the pyruvate kinase and L-lactate dehydrogenase genes.

A study was conducted to analyze the diversity of predominant bacteria in the human gastrointestinal tract. 16S rRNA-based approaches were utilized to carry out the analysis. Different profiles were determined using temperature gradient gel electrophoresis. Results indicated the effectiveness of the combination of cloning and temperature gradient gel electrophoresis analysis in evaluating rDNA amplicons and observing microbial communities in feces.

Lactococcus lactis bacterial strains are used in the production of dairy products worldwide and have the capacity to generate different end metabolites during sugar fermentation. Studies were conducted to modify metabolic flux in L. lactis on the level of NADH oxidation using metabolic engineering. Results of the research expand the insights derived on the pivotal function of redox balance on pyruvate metabolism and detail the use of co-factor in diacetyl overproduction.

Research was conducted to examine the regulated activity of the lactococcal nisA promoter in strains of gram-positive species Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacillus subtilis and Enterococcus faecalis. Results reveal that the lactococcal nisA promoter is recognized in many different gram-positive species which include pathogenic streptococci. Evidence also demonstrates that it was the most efficient of the regulatable promoters studied in B. subtilis and S. pyogenes.

A research study was conducted regarding the Carnobacterium piscicola (C piscicola) LV17B and its genetic requirement for bacteriocin production, necessary in minimizing food spoilage and growth of pathogenic bacteria in food. The methods used were the detection of bacteriocin production, analysis of bacteriocin sensitivity and manipulations of the bacterium's DNA and RNA. Results revealed that two endogenous quorum-sensing signals responsible for carnobacteriocin production are present in the C piscicola LV17B which also produces several bacteriocins.

A study was conducted on the impact of mannosylglycerate on the protection against freeze-drying of enzymes taken from thermophilic, mesophilic and hyperthermophilic sources. Results show that 2-0-Besta-mannosylglycerate, which accumulates in certain thermophilic organisms, is effective in stabilizing enzymes. Results also indicate the potential biotechnological applications of the solute.

The combination of the Escherichia coli gusA marker gene and the Pyrococcus furiosus celB gene helps in simultaneously detecting different Rhizobium strains on a plant and the presence of multiple-strains in individual nodules. The gusA gene marked strains and the celB gene marked strains help in the analysis of the whole system of roots, without analyzing the nodules. The celB gene is effective as a marker, since it encodes beta-glucosidase and the beta-galactosidase that are easily inactivated.

Sulfidogenic anaerobic granular sludge from aerobic sludge beds contains mainly the propionate-oxidizing bacteria Desulfobulbus sp. while methanogenic sludge contains mainly SYN7 bacteria. The Desulfobulbus content decreases with culturing in methanogenic sludge and increases in sulfidogenic sludge. SYN7 shows an opposite population dynamic. Desulfobulbus forms an outer thick layer on sulfidogenic sludge below which are microcolonies of methanogens.

Fluorescently labeled probes are used together with 16S rRNA-based probes for Bacteria and Archaea to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors fed with either sucrose or a mixture of volatile fatty acids. Results suggest that the hybridization of granular sludge with fluorescent probes is a powerful tool for studying structure-function relationships in situ.

Stable and flexible vectors based on the high-copy-number endogenous Lactococcus lactis (L. lactis) pSH71 replicon, the lacF selection marker and the lactose-inducible lacA promoter have been developed. The nucleotide sequences of the vectors consist entirely of L. lactis DNA, suggesting that the vectors can be applied to lactococci improvement through self-cloning. This would improve lactococci's role as hosts for protein/peptide/metabolite production for the food industry.

The transcription and translation initiation sites of the celB gene that encodes the enzyme beta-glucosidase from Pyrococcus furiosus are analyzed together with its expression and site-directed mutations in Escherichia coli. Results show that the alignment of the primary sequence of the celB gene with other members of the glycosyl hydrolase family has a large number of conserved amino acids. The data also suggest that the beta-glucosidase produce by E. coli via celB has kinetic and stability properties which are similar to those of beta-glucosidase from P. furiosus.

An overproduction of alpha-acetolactate synthase in Lactococcus (L.) lactis, coupled with optimum pH and aeration for fermentation, results in an 85% increase in the conversion of lactose to acetoin. The enzyme overproduction leads to butanediol formation which is 40% of the converted pyruvate in a dehydrogenase-deficient strain of L. lactis under anaerobic conditions. The enzyme overproduction is the result of the overexpression of the als gene.

The antimicrobial peptide nisin was modified to increase its solubility and stability. The solubility of nisins A and Z was greater at low ionic strength and low pH. The addition of lysyl residues to nisin Z increased its solubility at neutral pH without significantly affecting its antimicrobial activity. Replacing dehydroalanine on nisin Z with dehydrobutyrine resulted in increased resistance to acid-catalyzed degradation.