In the past decade much has been learnt about the order Cytophagales and about some of the organisms belonging to it, although the majority of its members are as unfamiliar as ever. On the basis of 16S rRNA studies, we can now delimit the group with some confidence and have a well-founded idea of its phylogenetic position (Paster et al., 1985; Woese et al., 1985). Accordingly, the Cytophagales appear to be distantly related to the Bacteroides group, and these two together comprise one of the main branches, perhaps a phylum, in the bacterial phylogenetic system. The substructure of the Cytophaga branch of the phylum is more difficult to evaluate. There is a main line on which we find unicellular gliders—Cytophaga (Cy.*) johnsonae, Cy. lytica, Cy. aquatilis = Flavobacterium (Fv.) aquatile, and Sporocytophaga (Sp.) myxococcoides—but at a lower level unicellular nonmotile bacteria (Fv. breve, i.e., low GC, true flavobacteria) are also found. At a still lower level, a cluster branches off which comprises the unicellular gliders—Flexibacter (Fx.) filiformis = Fx. elegans Fx el, Cy. heparina, and Taxeobacter = Myx 2105), unicellular non-gliding flavobacteria (Fv. ferugineum), but also filamentous, multicellular, gliding (Saprospira) and nonmotile bacteria (Haliscomenobacter). It is obvious from these data that our present definition of genera does not reflect the phylogenetic situation, and also that the grouping in families and perhaps orders needs to be reconsidered. Before that is done, however, 16S rRNA sequences of further species should be determined.

The Order Cytophagales

Chitin is the (1→4)-β-linked homopolymer of N-acetyl-D-glucosamine (Fig. 1). The individual polymer chains can be thought of as helices, as each sugar unit is inverted with respect to its neighbors. This leads to their stabilization as rigid ribbons by 03—H … 05 and 06—H … 07 hydrogen bonds. The commonest form of chitin is α-chitin. Its unit cell is of two N,N‵-diacetylchitobiose units of two chains in an antiparallel arrangement. Thus, adjacent polymer chains run in opposite directions, held together by 06—H … 06 hydrogen bonds, and the chains are held in sheets by 07 … H—N hydrogen bonds (Minke and Blackwell, 1978). This gives a statistical mixture of CH2OH orientations, equivalent to half oxygens on each residue, each forming inter- and intramolecular hydrogen bonds. This results in two types of amide groups; all are involved in the interchain C=O … H—N bonds, while half of the groups also serve as acceptors for 06—H … O=C intramolecular bonds. This extensive intermolecular hydrogen bonding leads to a very stable structure, the individual polymer chains eventually giving rise to microfibrils if allowed to crystallize (Gooday, 1983). A less common form of chitin is β-chitin, in which the unit cell is of one N,N‵diacetylchitobiose unit, giving a polymer stabilized as a rigid ribbon, as for α-chitin, by 03—H … 05 intramolecular bonds (Gardner and Blackwell, 1975). Chains are then held together in sheets by C=O … H—N hydrogen bonding of the amide groups and by the CH2OH side chains, forming intersheet hydrogen bonds to the carbonyl oxygens on the next chains (06—H … 07). This gives a structure of parallel poly-N-acetylglucosamine chains with no intersheet hydrogen bonds.

The Ecology of Chitin Degradation

The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the "motor" and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae.

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Novel Features of the Polysaccharide-Digesting Gliding Bacterium Flavobacterium johnsoniae as Revealed by Genome Sequence Analysis

The high affinity of Acinetobacter calcoaceticus RAG-1 for liquid hydrocarbons permitted the isolation of a spontaneous nonadherent mutant, MR-481. Strain MR-481 exhibited no significant affinity for three test hydrocarbons, yet resembled the wild type in many properties, including production of the extracellular emulsifying agent emulsan. To study the role of adherence in growth on hydrocarbons, RAG-1 and MR-481 were compared for growth on hexadecane under conditions of limited agitation and at low initial cell densities. Adherent RAG-1 cells were able to grow rapidly under these conditions, whereas nonadherent MR-481 cells failed to grow for at least 54 h. However, the addition of emulsan either initially or at various times after inoculation enabled the nonadherent MR-481 cells to grow on hexadecane. Growth was not the result of reversion of MR-481 from nonadherent to adherent cells. The data demonstrate that adherence is a crucial factor in the growth of A. calcoaceticus RAG-1 on hexadecane in the absence of extracellular emulsification of the substrate. Images

Role of adherence in growth of Acinetobacter calcoaceticus RAG-1 on hexadecane.

A review on the factors influencing biohydrogen production from lactate: The key to unlocking enhanced dark fermentative processes.

Decomposition of chitin by Cytophaga johnsonii was investigated. Unlike other chitinolytic bacteria, some strains of C. johnsonii did not liberate chitinase extracellularly; instead the cells of such strains had need for close contact with the chitin particles in order to hydrolyze them. The cell-free extract of one of these strains, viz., C. johnsonii C35 did show presence of chitinase. The remaining strains liberated an extracellular chitinase and a chitobiase. The partially purified chitinase from the culture filtrates of C. johnsonii C31 was most active at pH 6.3–6.5 and at 40°C. Metabolism of N-acetylglucosamine, a product of chitin hydrolysis, by C. johnsonii C35 and C31 was investigated. The nature of some of the products formed and the steps involved in their transformations by the resting cells and the cell-free extracts suggested that N-acetylglucosamine first gets deacetylated to glucosamine before it is further oxidized to glucosaminic acid by these strains. Both strains were also able to dehydrogenate gluconate to 2-ketogluconate, the former being in all probability the product of deamination of glucosaminic acid.

Breakdown of chitin by Cytophaga johnsonii.

An extracellular endopolygalacturonate lyase of Cytophaga johnsonii was purified from the culture filtrate. It appeared to be homogeneous as judged by polyacrylamide gel electrophoresis at pH 8.6 as well as pH 4.3. The purified enzyme had a pH optimum around 9.0 and required Ca++ ions for its maximum activity. The apparent Kmfor polygalacturonic acid was found to be 0.22%. Both paper and column chromatography indicated formation and accumulation of an unsaturated monomer along with unsaturated di-, tri-, tetra- and pentamers from polygalacturonic acid by the enzyme action, indicating that the enzyme cleaved the substrate randomly in a non-hydrolytic manner. The glycosidic linkage next to the non-reducing end of polygalacturonic acid was not resistant to attack by this enzyme unlike in other known polygalacturonate lyases.

Endo-polygalacturonate lyase of Cytophaga johnsonii.

WA~r~G and W~RKMAN, for instance~ have shown that an analysis of the fermentation of glucose by the iron deficient cells of A. indologenes resulted in the high yields of formic and lactic acids, low carbon dioxide production and in a prevention of the formation of succinic acid and molecular hydrogen. PAPPE~mME~ and SF_~S~KAN have demonstrated that a decrease in the iron content of a glucose fermentation causes Clo~tridium perfringens to shift from the normal types of fermentation characterized bY the formation of acetic acid, butyric acid, alcohol, carbon dioxide and hydrogen to one of pure lactic acid type. Recently, the influence of iron on the pathways of metabolism, extent of population changes, antigenic contents and pathogenecity of .Brucella suis has been the subject of a critical study reported in the literature (WA~I~G et al.). The influence of low and high levels of iron on the products of glucose fermentation by Butyribaeterium rettgeri has also been the topic of a very recent communication by PI~n ct al. The purpose of the present paper is to report the results obtained with Clostridium lacto-acetophilum in the iron deficient medium containing either glucose or lactate-acetate as the carbon source and to indicate that a progressive deficiency in iron has the effect of interfering with the normal growth of the bacterium as well as on the rate of oxidation of lactate and consequential formation of butyrate, the typical metabolic product of this organism.

J. V. Bhat

Papers

Breakdown of chitin by Cytophaga johnsonii.

Endo-polygalacturonate lyase of Cytophaga johnsonii.

Role of iron in the nutrition and metabolism of clostridium lacto-acetophilum