Bdellovibrio in methylotrophic bacteria

Summary During the production of single cell protein ofMethylomonas sp. using methanol as the sole carbon source in the pilot plant scale, we isolated aBdellovibrio strain from an abnormal fermentation broth. The abnormality of fermentation caused byBdellovibrio was much like phage infection. However, the plaques formed byBdellovibrio enlarged progressively when plated with host.     

Peptidoglycan in obligate intracellular bacteria

Peptidoglycan is the predominant stress‐bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted ‘peptidoglycan‐intermediate’ organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan‐negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan‐intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan‐like sacculus and/or division septum.     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

Nitrogen assimilation in facultative methylotrophic bacteria

A study was done of the pathways of nitrogen assimilation in the facultative methylotrophsPseudomonas MA andPseudomonas AM1, with ammonia or methylamine as nitrogen sources and with methylamine or succinate as carbon sources. When methylamine was the sole carbon and/or nitrogen source, both organisms possessed enzymes of the glutamine synthetase/glutamate synthase pathway, but when ammonia was the nitrogen sourcePseudomonas AM1 also synthesized glutamate dehydrogenase with a pH optimum of 9.0, andPseudomonas MA elaborated both glutamate dehydrogenase (pH optimum 7.5) and alanine dehydrogenase (pH optimum 9.0). Glutamate dehydrogenase and glutamate synthase from both organisms were solely NADPH-dependent; alanine dehydrogenase was NADH-dependent. No evidence was obtained for regulation of glutamine synthetase by adenylylation in either organism, nor did glutamine synthetase appear to regulate glutamate dehydrogenase synthesis.     

Carbohydrate metabolism in some methylotrophic bacteria

Abstract Some pink pigmented facultative methylotrophic bacteria (PPFMs) can utilize monosaccharides as a single carbon source. Assays of key enzymes of various pathways of carbohydrate metabolism indicate that such strains either metabolise glucose by the Entner-Doudoroff pathway or lack a suitable permease for this sugar.     

Genetics of carbon metabolism in methylotrophic bacteria

Abstract The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer ( Paracoccus denitrificans ) and three methanotrophs ( Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C₁ genes has been conserved to some extent in methylotrophic bacteria, and that many C₁ genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.     

Mobile DNA in obligate intracellular bacteria

The small genomes of obligate intracellular bacteria are often presumed to be impervious to mobile DNA and the fluid genetic processes that drive diversification in free-living bacteria. Categorized by reductive evolution and streamlining, the genomes of some obligate intracellular bacteria manifest striking degrees of stability and gene synteny. However, recent findings from complete genome sequences of obligate intracellular species and their mobile genetic associates favour the abandonment of these wholesale terms for a more complex and tantalizing picture.     

Physiology and genetics of methylotrophic bacteria

Methylotrophic bacteria comprise a broad range of obligate aerobic microorganisms, which are able to proliferate on (a number of) compounds lacking carbon-carbon bonds. This contribution will essentially be limited to those organisms that are able to utilize methanol and will cover the physiological, biochemical and genetic aspects of this still diverse group of organisms. In recent years much progress has been made in the biochemical and genetic characterization of pathways and the knowledge of specific reactions involved in methanol catabolism. Only a few of the genetic loci hitherto found have been matched by biochemical experiments through the isolation or demonstration of specific gene products. Conversely, several factors have been identified by biochemical means and were shown to be involved in the methanol dehydrogenase reaction or subsequent electron transfer. For the majority of these components, their genetic loci are unknown. A comprehensive treatise on the regulation and molecular mechanism of methanol oxidation is therefore presented, followed by the data that have become available through the use of genetic analysis. The assemblage of methanol dehydrogenase enzyme, the role of pyrrolo-quinoline quinone, the involvement of accessory factors, the evident translocation of all these components to the periplasm and the dedicated link to the electron transport chain are now accepted and well studied phenomena in a few selected facultative methylotrophs. Metabolic regulation of gene expression, efficiency of energy conservation and the question whether universal rules apply to methylotrophs in general, have so far been given less attention. In order to expand these studies to less well known methylotrophic species initial results concerning such area as genetic mapping, the molecular characterization of specific genes and extrachromosomal genetics will also pass in review.     

The taxonomy of obligate anaerobic bacteria

Studies on the chemotaxonomy of obligate anaerobic bacteria have been made. The combination of gas chromatography and mass spectrometry with computer-assisted analysis, permitting the multicomponent analysis of all products of bacterial metabolism and bacterial cell components, has been shown to be a research method, quite suitable for such studies. The chromatographic profiles of the end products of metabolism in anaerobic cultures of different age have been found to differ not in the set and number of peaks indicating various metabolites, but only in the concentration of metabolites, increasing in the process of prolonged incubation. The authors believe that the national microbiological "library" of the chromatographic profiles of anaerobic organisms should be created and the album of typing chromatographic profiles should be published; besides, data on new profiles should regularly appear in magazines.     

Development of a system vector-host in methylotrophic bacteria

Cloning of methylotropic and other Gram negative bacteria's genes was performed using vectors derived from IncP4 plasmids. Plasmids, such s RSF1010 are 8.8 kb in length, have a high copy number and broad host range and can be mobilized efficiently by a number of conjugative plasmids. IncP4 plasmids have relatively few restriction enzyme's targets suitable for cloning. In this paper the construction of versatile and special purpose IncP4 vectors available for cloning DNA into broad range of bacterial species are described. The seria of versatile vectors involves the transposon containing plasmid and two-replicon vectors.In genetic construction of special vector for direct cloning of restriction fragments the genetic regulation elements of Tn 1 were used. On the base of IncP4 replicon special vectors for construction of bank genes (cosmids) and the vectors for cloning of regulation sequence were also constructed.     

Regulation of citrate synthase in facultative methylotrophic bacteria

Commonly the TCA cycle fulfils an anabolic and a catabolic function in case of aerobic chemoorganoheterotrophic nutrition. In methylotrophic growth the TCA cycle is dispensable as a bioenergetic pathway. This is reflected by properties of citrate synthase in facultative methylotrophic bacteria. Two citrate synthases, a "chemoorganoheterotrophic" one, which is inhibited by NADH (or ATP in Acetobacter MB 58), and a "methylotrophic" one, which is not or less affected by energy indicators, were found in Pseudomonas oleovorans, Pseudomonas MS, Pseudomonas MA, and Acetobacter MB 58. The concentration of these citrate synthases depends on the manner of nutrition. Bacteria with ICL-negative-variant of the serine pathway and with ribulosebisphosphate pathway seem to possess only a "chemoorganoheterotrophic" citrate synthase. Possibly the anabolic function of this citrate synthase can be realized by metabolites.     

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

An obligate methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment

A new, obligately methylotrophic, methane-oxidizing bacterium, strain AMO 1, was isolated from a mixed sample of sediments from five highly alkaline soda lakes (Kenya). Based on its cell ultrastructure and high activity of the hexulose-6-phosphate synthase, the new isolate belongs to the type I methanotrophs. It differed, however, from the known neutrophilic methanotrophs by the ability to grow and oxidize methane at high pH values. The bacterium grew optimally with methane at pH 9–10. The oxidation of methane, methanol, and formaldehyde was optimal at pH 10, and cells were still active up to pH 11. AMO 1 was able to oxidize ammonia to nitrite at high pH. A maximal production of nitrite from ammonia in batch cultures at pH 10 was observed with 10% of CH₄ in the gas phase when nitrate was present as nitrogen source. Washed cells of AMO 1 oxidized ammonia most actively at pH 10–10.5 in the presence of limiting amounts of methanol or CH₄. The bacterium was also capable of oxidizing organic sulfur compounds at high pH. Washed cells grown with methane exhibited high activity of CS₂ oxidation and low, but detectable, levels of DMS and DMDS oxidation. The GC content of AMO 1 was 50.9 mol%. It showed only weak DNA homology with the previously described alkaliphilic methanotroph "Methylobacter alcaliphilus" strain 20 Z and with the neutrophilic species of the genera Methylobacter and Methylomonas. According to the 16S rRNA gene sequence analysis, strain AMO 1 was most closely related to a neutrophilic methanotroph, Methylomicrobium pelagicum (98.2% sequence similarity), within the gamma-Proteobacteria. Received: July 26, 1999 / Accepted: January 4, 2000