Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60%o of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis.Alcaligenes eutrophus H16 is a facultative lithoautotroph classified in the ,-purple group of gram-negative bacteria. In the absence of organic substrates, A. eutrophus can grow on a mixture of CO2, H2, and 02 Under these conditions, carbon dioxide is fixed via the reductive pentose phosphate (Calvin) cycle (5). The enzymes which catalyze CO2 fixation are encoded by the cbb (formerly cfx) genes. Two copies of these genes are present: one is located on the bacterial chromosome, and the other is carried on the 450-kb conjugative megaplasmid pHG1 (21,23,26,49). The central energy-yielding process in the lithoautotrophic metabolism of A. eutrophus is the oxidation of molecular hydrogen. This reaction is catalyzed by two enzymes: a heterotetrameric, NAD-reducing hydrogenase (SH) found in the cytoplasm and a heterodimeric hydrogenase (MBH) which is attached to the cytoplasmic membrane (16,33,37,38). Both enzymes are nickel metalloproteins (11). The two hydrogenases are encoded in separate operons located in the hox gene cluster of pHG1 (25,46). The formation of the hydrogen-oxidizing apparatus requires, in addition to the hydrogenase enzymes, a set of accessory proteins instrumental to the maturation of the hydrogenase polypeptides, to nickel uptake, and to the insertion of nickel into the active sites of the enzymes. These accessory proteins are also encoded by genes of the megaplasmid hox cluster (6-8, 10, 24, 25).Both the synthesis of the Calvin cycle enzymes and the formation of the hydrogen-oxidizing apparatus are regulated efficiently (13,27). During growth on preferentially utilized organic substrates, both enzyme systems are tightly repressed; derepression takes place upon transition to lithoautotrophic conditions. However, the two systems are controlled by separate regulatory circuits. The cbb genes are regulated by the product of the cbbR gene, a member of the LysR family of bacterial regulatory proteins (50). Expression of the hox genes is governed by the product of the hoxA locus. Protein HoxA is a member of the ...

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH- and MBH-specific antigen. Growing the cells in the presence of 63Ni2+ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.

The transfer of extended spectrum β-lactamase (ESBL)-genes occurs frequently between different bacteria species. The aim of this study was to investigate the impact of nutrition related stress factors on this transfer. Thus, an Escherichia coli donor and a Salmonella Typhimurium recipient were co-incubated for 4 h in media containing different levels of the stress factors’ pH, osmolality, copper, zinc and acetic, propionic, lactic, and n-butyric acid, as well as subtherapeutic levels of cefotaxime, sulfamethoxazole/trimethoprim, and nitrofurantoin. Conjugation frequencies were calculated as transconjugants per donor, recipient, and total bacterial count. A correction factor for the stress impact on bacterial growth was used. Acetic, lactic, and n-butyric, acid, as well as pH, showed no significant impact. In contrast, increasing concentrations of propionate, zinc, copper, and nitrofurantoin, as well as increased osmolality reduced conjugation frequencies. Sulfamethoxazole/trimethoprim and cefotaxime showed increased transconjugants per donor, which decreased after correction for stress. This study showed, for the model mating pair, that conjugation frequencies decreased under different physiological stress conditions, and, thus, the hypothesis that stress factors may enhance conjugation should be viewed with caution. Furthermore, for studies on in vitro gene transfer, it is vital to consider the impact of studied stressors on bacterial growth.

The nucleotide sequence of the rpoN gene, formerly designated hno, and flanking DNA regions of the aerobic hydrogen bacterium Alcaligenes eutrophus has been determined; rpoN codes for the RNA polymerase sigma factor sigma 54 involved in nitrogen regulation and diverse physiological functions of gram-negative bacteria. In A. eutrophus hydrogen metabolism is under control of rpoN. The Tn5-Mob insertion in a previously isolated pleiotropic mutant was mapped within the rpoN gene. The derived amino acid sequence of the A. eutrophus RpoN protein shows extensive homology to the RpoN proteins of other organisms. Sequencing revealed four other open reading frames: one upstream (ORF280) and three downstream (ORF130, ORF99 and ORF greater than 54) of the rpoN gene. A similar arrangement of homologous ORFs is found in the rpoN regions of other bacteria and is indicative of a conserved gene cluster.