- Volume 147, Issue 1, 2001

Psudomonas putida CE2010 can assimilate biphenyl despite its high similarity to P. putida F1. Biphenyl degradation in strain CE2010 was achieved using a mosaic of pathways consisting of the cmt and tod operons. CmtE hydrolysed 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage product of 2,3-dihydroxybiphenyl. This enzyme was expressed differently in strains CE2010 and F1. A cmtE disruption mutant, a tod operon disruption mutant and a cmt operon disruption mutant were unable to utilize biphenyl. The introduction of the cmtE gene enabled the cmt operon disruption mutant to grow on biphenyl. A single base difference was found in the cmt promoter–operator region in strain CE2010, compared with that of strain F1. CymR protein was purified from Escherichia coli and binding assays were performed, the results of which suggested that the protein bound less strongly to the CE2010 operator sequence than to the F1 operator sequence. Exchanging the F1 promoter–operator fragment into strain CE2010 resulted in a loss of biphenyl degradation capacity. These results indicate that cmtE is not effectively repressed by CymR in strain CE2010, leading to low constitutive expression and, therefore, low growth on biphenyl.

ΔD transformants containing all 14 gvp genes of Haloferax mediterranei required for gas vesicle formation except for gvpD are gas vesicle overproducers (Vac++), whereas ΔD/D transformants containing the gvpD reading frame under ferredoxin promoter control on a second construct in addition to ΔD did not form gas vesicles (Vac−). The amino acid sequence of GvpD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by mutation, and the resulting GvpDmut proteins tested in ΔD/Dmut transformants for their ability to repress gas vesicle formation. The exchange of amino acids at conserved positions in the p-loop motif resulted in Vac++ ΔD/Dmut transformants, indicating that these GvpDmut proteins were unable to repress gas vesicle formation. In contrast, a GvpDmut protein with an alteration of a non-conserved proline in the p-loop region (P41A) was still able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gvpA gene. This promoter is only activated during the stationary phase, depending on the transcriptional activator protein GvpE. Whereas the Vac++ ΔD transformants contained very high amounts of gvpA mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac− transformants ΔD/D and ΔD/DP41A. In contrast, the Vac++ ΔD/Dmut transformants harbouring GvpDmut with mutations at conserved positions in the p-loop motif contained large amounts of gvpA mRNA already during exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing mutations in the two basic regions were mostly defective in the repressing function. The GvpDmut protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle formation. No gvpA mRNA was detectable in this transformant, demonstrating that this GvpD protein was acting as a strong repressor. All these results imply that the GvpD protein is able to prevent the GvpE-mediated gvpA promoter activation, and that the p-loop motif as well as the two basic regions are important for this function.

Clostridium acetobutylicum DSM 792 accumulates and phosphorylates mannitol via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). PEP-dependent mannitol phosphorylation by extracts of cells grown on mannitol required both soluble and membrane fractions. Neither the soluble nor the membrane fraction could be complemented by the opposite fraction prepared from glucose-grown cells, indicating that the mannitol-specific PTS consists of both a soluble (IIA) and a membrane-bound (IICB) component. The mannitol (mtl) operon of C. acetobutylicum DSM 792 comprises four genes in the order mtlARFD. Sequence analysis of deduced protein products indicated that the mtlA and mtlF genes respectively encode the IICB and IIA components of the mannitol PTS, which is a member of the fructose-mannitol (Fru) family. The mtlD gene product is a mannitol-1-phosphate dehydrogenase, while mtlR encodes a putative transcriptional regulator. MtlR contains two PTS regulatory domains (PRDs), which have been found in a number of DNA-binding transcriptional regulators and in transcriptional antiterminators of the Escherichia coli BglG family. Also, near the C-terminus is a well-conserved signature motif characteristic of members of the IIAFru/IIAMtl/IIANtr PTS protein family. These regions are probably the sites of PTS-dependent phosphorylation to regulate the activity of the protein. A helix–turn–helix DNA-binding motif was not found in MtlR. Transcriptional analysis of the mtl genes by Northern blotting indicated that the genes were transcribed as a polycistronic operon, expression of which was induced by mannitol and repressed by glucose. Primer extension experiments identified a transcriptional start point 42 bp upstream of the mtlA start codon. Two catabolite-responsive elements (CREs), one of which overlapped the putative −35 region of the promoter, were located within the 100 bp upstream of the start codon. These sequences may be involved in regulation of expression of the operon.

Burkholderia sp. NK8 grows abundantly on 3-chlorobenzoate (3CB),4-chlorobenzoate (4CB) and benzoate. The genes encoding the oxidation of (chloro)benzoates (cbeABCD) and catechol (catA, catBC), the LysR-type regulatory gene cbeR and the gene cbeE with unknown function, all of which form a single cluster in NK8, were cloned and analysed. The protein sequence of chlorobenzoate 1,2-dioxygenase (CbeABC) is 50–65% identical to the benzoate dioxygenase (BenABC) of Acinetobacter sp. ADP1, toluate dioxygenase (XylXYZ) of the TOL plasmid pWW0 and 2-halobenzoate dioxygenase (CbdABC) of Burkholderia cepacia 2CBS. Disruption of the cbeA gene resulted in the simultaneous loss of the ability to grow on benzoate and monochlorobenzoates, indicating the involvement of the cbeABCD genes in the degradation of these aromatics. The cbeABCD genes are preceded by catA, the gene for catechol dioxygenase. lacZ transcriptional fusion studies in Pseudomonas putida showed that catA and cbeA are co-expressed under the positive control of cbeR, a LysR-type transcriptional regulatory gene. The cbeA::lacZ transcriptional fusion studies showed that the inducers of the genes are 3CB, 4CB, benzoate and probably cis,cis-muconate. On the other hand, 2-chlorobenzoate (2CB) did not activate the expression of the genes. The chlorobenzoate dioxygenase was able to transform 2CB, 3CB, 4CB and benzoate at considerable rates. 2CB yielded both catechol and 3-chlorocatechol (3CC), and 3CB gave rise to 4-chlorocatechol and 3CC as the major and minor intermediate products, respectively, indicating that the NK8 dioxygenase lacks absolute regiospecificity. The absence of growth of NK8 on 2CB, despite its considerable degradation activity against 2CB, is apparently due to the inability of CbeR to recognize 2CB as an inducer of the expression of the cbe genes.

Previous research has shown that verotoxin-producing Escherichia coli (VTEC) O157 strains appear unable to express type 1 fimbriae although other serotypes such as O26 and O118 can. This study has investigated the molecular basis of this difference. The study confirmed the presence of a 16 bp deletion within the regulatory region of fimA (fim switch) in 63 VTEC O157 strains but not in other VTEC serotypes tested. The fim switch was shown to be detectable only in the phase off orientation in VTEC O157, but detection of the switch in the phase on orientation correlated with the degree of mannose-sensitive yeast agglutination in VTEC O26. Repair of the 16 bp deletion in the VTEC O157 fim switch region restored phase-variable expression of fimA in a permissive background. Non-O157 VTEC, especially O26 and O118, can be pathogenic in cattle; the role of type 1 fimbriae in this and colonization is discussed.

A binding site for DnaA protein was identified in the regulatory region of the aldA gene of Escherichia coli. Specific binding was demonstrated by in vitro assays including filter binding as well as mobility shift in a polyacrylamide gel of the DnaA–DNA complex. In cells growing in minimal medium containing glucose, expression of β-galactosidase activity from an aldA–lacZ fusion gene was suppressed by oversupply of DnaA protein and was enhanced by reducing the free DnaA level. These results suggest that DnaA protein negatively regulates expression of the aldA gene under these conditions. Despite fairly strong binding, the bound DNA fragment had no consensus 9 bp DnaA binding sequence (DnaA box), and anomalous binding to an AT-rich sequence located close to the transcription start site was revealed by a footprinting experiment.

The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans. In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes. However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown. Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene. This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication. These defects in diverse cellular processes found in the insertional cgtA mutant of V. harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of \(NH_{4}^{+}\) or removal from light, resulting in a decrease in nitrogenase activity. DRAT is itself subject to post-translational regulation; to investigate the mechanism for the regulation of DRAT activity, random PCR mutagenesis of draT (encoding DRAT) was performed and mutants with altered DRAT regulation were screened. Two mutants (with substitutions of K103E and N248D) were obtained in which DRAT showed activity under conditions where wild-type DRAT (DRAT-WT) did not. These mutants showed lower nitrogenase activity and a higher degree of ADP-ribosylation of dinitrogenase reductase under N2-fixing conditions than was seen in a wild-type control strain. DRAT-K103E was overexpressed and purified. DRAT-K103E displayed a much weaker affinity for an Affi-gel Blue matrix than did DRAT-WT, suggestive of a fairly striking biochemical change. However, there was no significant difference in kinetic constants, such as K m for NAD and V max, between DRAT-K103E and DRAT-WT. Like DRAT-WT, DRAT-K103E also modified reduced dinitrogenase reductase poorly. The biochemical properties of these variants are rationalized with respect to their behaviour in vivo.

The gene encoding periplasmic 2′,3′-cyclic phosphodiesterase in Yersinia enterocolitica O:8 (designated cpdB), was cloned and expressed in Escherichia coli. This enzyme enables Y. enterocolitica to grow on 2′,3′-cAMP as a sole source of carbon and energy. Sequencing and analysis of a 3 kb EcoRI fragment containing the cpdB gene revealed an open reading frame of 1179 bp, corresponding to a protein with a molecular mass of 71 kDa. The first 25 amino acid residues show features of a typical prokaryotic signal sequence. The predicted molecular mass of the mature peptide is therefore in agreement with the molecular mass estimated by SDS gel electrophoresis (68 kDa). The putative cpdB promoter region contains two possible −10 and −35 regions. Furthermore, the 5′ untranslated region contains sequences with significant homology to the cyclic AMP–cyclic AMP receptor protein binding site and the σ28 consensus. This region is interrupted by an enterobacterial repetitive intergenic consensus (ERIC) sequence. Deletion of the ERIC element from the cpdB promoter region had no effect on cpdB expression. In the 3′ untranslated region, a possible rho-independent transcriptional terminator was identified. The deduced amino acid sequence of the Y. enterocolitica CpdB protein shows 76% identity with CpdB of Salmonella typhimurium and E. coli. CpdB of Y. enterocolitica is exported to the periplasmic space. An isogenic Y. enterocolitica cpdB mutant strain, constructed by allelic exchange, was no longer able to grow on 2′,3′-cAMP as sole source of carbon and energy. The CpdB mutant showed no significant change in virulence in an oral and intravenous mouse infection model.

Amino acid aminotransferases (ATases), which catalyse the last biosynthetic step of many amino acids, may have important physiological functions in Lactococcus lactis during growth in milk. In this study, the aspartate ATase gene (aspC) from L. lactis LM0230 was cloned by complementation into Escherichia coli DL39. One chromosomal fragment putatively encoding aspC was partially sequenced. A 1179 bp ORF was identified which could encode for a 393 aa, 43·2 kDa protein. The deduced amino acid sequence had high identity to other AspC sequences in GenBank and is a member of the Iγ family of ATases. Substrate-specificity studies suggested that the lactococcal AspC has ATase activity only with aspartic acid (Asp). An internal deletion was introduced into the L. lactis chromosomal copy of aspC by homologous recombination. The wild-type and mutant strain grew similarly in defined media containing all 20 amino acids and did not grow in minimal media unless supplemented with asparagine (Asn). The mutant strain was also unable to grow in or significantly acidify milk unless supplemented with Asp or Asn. These results suggest that only one lactococcal ATase is involved in the conversion of oxaloacetate to Asp, and Asp biosynthesis is required for the growth of L. lactis LM0230 in milk.

The minCDE genes involved in division site selection in Neisseria gonorrhoeae were identified using raw data from the N. gonorrhoeae genome project and are part of a cluster of 27 genes. When gonococcal min genes were heterologously expressed as a cluster in Escherichia coli, minicells and filaments were produced, indicating that gonococcal min genes disrupted cell division in other genera. The insertional inactivation of the minC gene of N. gonorrhoeae CH811 resulted in a strain (CSRC1) with decreased viability and grossly abnormal cell division as observed by phase-contrast and electron microscopy analysis. Western blot analysis of N. gonorrhoeae CSRC1 confirmed that MinCNg was not produced. Complementation of CSRC1 by integrating a minC–6×His tag fusion at the proAB locus by homologous recombination restored viability and 1·9 times wild-type levels of MinCNg expression. This slight increase of expression caused a small percentage of the complemented cells to divide aberrantly. This suggested that the 6×His tag has partially affected the stability of MinC, or that the chromosomal position of minC is critical to its regulation. Comparison of MinC proteins from different bacteria showed a homologous region corresponding to residues 135–230 with five conserved amino acids. Overexpression of MinCNg in wild-type E. coli cells induced filamentation and an E. coli minC mutant was successfully complemented with minC Ng. Therefore, the evidence indicates that MinC from N. gonorrhoeae acts as a cell-division inhibitor and that its role is essential in maintaining proper division in cocci.